Anal. Chem. 1996, 68, 3483-3488
Application of Compact Porous Disks for Fast Separations of Biopolymers and In-Process Control in Biotechnology A. S ˇ trancar and P. Koselj
BIA, d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia Horst Schwinn and Djuro Josic*
Octapharma Pharmazeutika Produktionsges. m.b.H., Oberlaaer Strasse 235, A-1100 Wein, Austria
Production and downstream processing in biotechnology requires fast and accurate control of each step in the process. Improved techniques which can be validated are required in order to meet these demands. For these purposes, chromatographic units containing compact porous disks for fast separation of biopolymers were developed and investigated with regard to their performance and speed. The problems that have, in the past, arisen from the use of wide and flat separation units, such as membranes and disks, have chiefly been those of sample distribution and large void volumes before and behind the unit. Improvements in the construction of the cartridge have led to better performance of the compact porous disks and faster separation. Using these disks, three calibration standard proteins could be separated within less than 1 min by an anion-exchange, cationexchange, and hydrophobic interaction mode. Such units can be used for in-process control in production and downstream processing of biopolymers, as was shown in experiments involving the purification of r1-antitrypsin and clotting factor IX and the immobilization of enzyme glucose oxidase on an epoxy-activated compact porous disk. Monitoring of production processes in biotechnology is a basic requirement for optimization. Exact data are needed, not only to optimize the processes themselves, but also to satisfy the demands of regulatory authorities with regard to subsequent registration of the product as a drug. Analytical methods such as gas chromatography (GC), capillary electrophoresis (CE), and especially high-performance liquid chromatography (HPLC) allow monitoring of one or more key substances without interference in the process itself. Recently, dramatic progress has been seen in HPLC of biopolymers, especially in a reduced-time of separation and an improved separation performance. It is to be anticipated that this analytical method will be among the methods of choice where on-line monitoring is concerned.1,2 Sensors have recently been discussed as a possible replacement for chromatographic methods for on-line monitoring.3 So (1) Csen, H.; Horvath, Cs. J. Chromatogr. A 1995, 705, 3-20. (2) Paliwal, S. K.; Nadler, T. K.; Wang, D. J. C.; Regnier, F. E. Anal. Chem. 1993, 65,3363-3367. S0003-2700(96)00292-2 CCC: $12.00
© 1996 American Chemical Society
far, as far their capability is concerned, they are generally inferior to HPLC, especially when fast and quantitative analysis of several components of the sample is needed.1 In the past few years, different approaches have been made to monitor fermentation processes, or different downstream processing operations, in biotechnology by fast HPLC or by similar fast chromatographic methods.2,4-6 Also, efforts have been made to improve the continuous sampling of complicated production processes in biotechnology. In-line and on-line devices have been devised for the removal of solid particles, agglomerates, and cells from the sample in order both to obtain reliable results and to extend the life of the analytical unit.5,7,8 Unfortunately, many problems, e.g., relatively large dead volumes of the devices and short working life due to their blockage, have not yet been solved satisfactorily. The appearance of nonporous, so-called micropellicular stationary phases was a decisive breakthrough for in-process control by allowing the development of fast HPLC of biolpolymers.9-12 Subsequent developments, namely “perfusion packings” 13,14 and other “fast” separation media,15,16 have provided further progress in this area. Owing to their fast mass transfer, these supports permit separations as rapid as those carried out on micropellicular stationary phases. Other advantages of these porous stationary phases over nonporous stationary phases are lower back pressure of the column; higher capacity, even with faster flow rates, than that of the nonporous stationary phases;14-16 and possible applica(3) Reilly, M. T.; Charles, M.; Twork, J. V.; Yacynych, A. M.The Use of OnLine Sensors in Bioprocess Control In Sensors in Bioprocess Control Twork, J. V., Yacynych, A. M., Eds.; Bioprocess Technology 6; Marcel Dekker, Inc.: New York, 1990; pp 243-292. (4) Lundstrom, H.; Brohjer, M.; Osterlof, B.; Moks, T. Biotechnol. Bioeng. 1990, 36, 1056-1062. (5) Reif, O. W.; Freytag, R. J. Chromatogr. A 1993, 654, 29-41. (6) Josic, Dj.; Bal, F.; Schwinn, H. J. Chromatogr. 1993, 632, 1-10. (7) Schu ¨ gerl, K. Anal. Chem. 1988, 213, 1-9. (8) Nielsen, J. Process Control Qual. 1992, 2, 371-384. (9) Unger, K. K.; Jilge, G.; Kinkel, J. N.; Hearn, M. T. W. J. Chromatogr. 1986, 359, 61-72. (10) Burke, D. J.; Duncan, J. K.; Dunn, L. C.; Cummings, L.; Siebert, C. J.; Ott, G. S. J. Chromatogr. 1986, 353, 425-437. (11) Kalghatgi, K.; Horvath, C. S. J. Chromatogr. 1987, 398, 335-339. (12) Hashimoto, T. J. Chromatogr. 1991, 544, 257-265. (13) Fulton, S. P.; Afeyan, N. B.; Gordon, N. F.; Regnier, F. E. J. Chromatogr. 1991, 547, 452-456. (14) Afeyan, N. B.; Fulton, S. P.; Regnier, F. E. LC-GC 1991, 9, 824-832. (15) Gaganon, P.; Grund, E.; Lindba¨ck, T. BioPharm 1995, 8 (3), 21-27. (16) Horvath, J.; Boschetti, E.; Guerrier, L.; Cooke, N. J. Chromatogr. A 1994, 679, 11-22.
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tion for preparative purposes in downstream processing. Apart from optimization of supports, studies carried out to test the influence of other factors on column performance revealed that, for the separation of biopolymers using gradient elution, column performance depends to only a small extent on its length. It was also shown that separations can be carried out at much higher flow rates than had been previously assumed.17-19 These findings resulted in the introduction of short columns and fast flow rates for protein separations.1,11 Another consequence of these studies is the introduction of so-called high-performance membrane chromatography (HPMC). This method replaces the chromatographic column with one membrane or several “piled-up” membranes, made of regenerated cellulose or synthetic material.5,20,21 An alternate option is the use of compact disks made of hollow fibers or of a synthetic material such as poly(styrene-co-divinylbenzene) (PS-DVB) or poly[(glycidyl methacrylate)-co-(ethylene dimethacrylate)] (GMAEDMA).22-26 Both membranes and compact disks show marked similarities in their separation performance as compared to fast, so-called perfusion supports for the separation of biopolymers. The same speed was achieved, the capacity of the separation unit was high, and scaling-up with the same material was accomplished.21,25 A major problem in the use of short chromatographic columns is the formation of canals and the distribution of the sample at the inlet of the separation unit. If membranes or disks are used, the need to facilitate sample distribution has to be solved.24,25 In this report, the use of compact, porous disks for fast separation of biopolymers is demonstrated. The speed of separation and the reliability of the separation units permit their use for in-process analysis, as is shown in the experiments. MATERIALS AND METHODS Instrumentation. A gradient HPLC system was built with two HPLC 64 pumps, an injection valve with a 20 µL PEEK sample loop, a variable wavelength monitor with a 10 mm optical path, a 10 µL volume flow cell, and HPLC software/hardware (data acquisition and control station), connected by means of 0.25 mm i.d. PEEK capillary tubes (all from Knauer, Berlin, Germany). The equipment was used with some minor modifications in all analytical chromatographic runs. Knauer’s mixing chamber, with its relatively high dead volume, was replaced by the PEEK biocompatible mixing tree, with extra-low dead volume (Jour Research, Uppsala, Sweden). For protein immobilization on epoxy-activated disks, a lowpressure P-50 pump (Pharmacia, Wien, Austria) was used. Chemicals. Human serum albumin (HSA, purified) was obtained from Behring (Marburg, Germany). Myoglobin (Mb, (17) Stadalius, M. S.; Quarry, M. S.; Snyder, L. R. J. Chromatogr. 1985, 327, 93-113. (18) Dong, M. W.; Gant, J. R.; Larsen, B. R. Biochromatography 1989, 3, 19-25. (19) Kopaczewicz, W.; Kellard, E.; Cox, G. B. J. Chromatogr. A 1995, 690, 9-19. (20) Gerstner, J. A.; Hamilton, R.; Cramer, S. J. J. Chromatogr. 1992, 596, 173180. (21) Roper, D. K.; Lightfoot, E. N. J. Chromatogr. A 1995, 702, 3-26. (22) Tennikova, T. B.; Belenkii, B. G.; Svec, F. J. Liq. Chromatogr. 1990, 13, 63-70. (23) Abou-Rebyeh, H.; Ko ¨rber, F.; Schubert-Rehberg, K.; Reusch, J.; Josic, Dj. J. Chromatogr. Biomed. Appl. 1991, 566, 341-350. (24) Belenkii, B. G.; Malt’sev, V. G. BioTechniques 1995, 18 (2), 288-291. (25) Josic, Dj.; Reusch, J.; Lo ¨ster, K.; Baum, O.; Reutter, W. J. Chromatogr. 1992, 590, 59-76. (26) Svec, F.; Jelinkova, M.; Votavova, E. Angew. Makromol. Chem. 1991, 188, 167-176.
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from horse skeletal muscle, 95-100%), conalbumin (CA, from chicken egg white, 99%), trypsin inhibitor (STI, from soybean), transferrin (Tf, from human plasma, ∼98%), R1-antitrypsin (AAT, from human plasma), RNAse (from bovine pancreas, 85%), cytochrome c (Cytc), and lysozyme (Lys, from chicken egg white) were all purchased from Sigma Chemical Co. (St. Louis, MO). Clotting factor IX (FIX, from human plasma) was obtained from Octapharma (Wien, Austria). Mononine, another FIX preparation, was obtained from Armour Pharma GmbH (Eschwege, Germany). All chemicals were of analytical grade and purchased from E. Merck (Darmstadt, Germany). Eluents were prepared in the concentrations listed in the figures and figure legends. The water used for preparing eluents was twice distilled (Octapharma). When used with columns, eluents were filtered in an 0.45 µm filter (Millipore, Wien, Austria) and degassed on an ultrasound bath (Knauer) before use. Separation Devices. QA, DEAE, SO3, HIC (propyl-), and epoxy compact porous disks were synthesized in BIA d.o.o. (Ljubljana, Slovenia) by a radical copolymerization of glycidyl methacrylate and ethylene dimethacrylate in the presence of poreproducing solvents, following the method of Svec et al.26 Before the chemical modification was carried out, the disks were rinsed with methanol, a methanol-water mixture (1:1), and finally water to remove any residues of compounds that had failed to react. The chemical modifications of epoxy groups turned into ionexchange and hydrophobic interaction groups have been described elsewhere.26 Disks of 3 mm thickness were prepared with two different diameters, 10 and 25 mm. They were then mounted in a special cartridge with a low dead volume. The cartridges were made of poly(propylene) and developed by BIA d.o.o. Immobilization of Glucose Oxidase on Compact Disks. For protein immobilization, the ligand was positioned on the disk in epoxy form in situ, according to the protocol published elsewhere.25 After installing the disk (3 mm × 10 mm i.d.) in the appropriate cartridge, any remaining nonpolymerized components were washed out with 20 mL of methanol. The disk was then rinsed with twice-distilled water and 0.1 M sodium phosphate buffer, pH 7.5. In the case of a 10 mm diameter disk, thickness 3 mm, 3 mg of glucose oxidase (GOX, Boehringer, Mannheim, Germany) in 5 mL of 0.1 M sodium phosphate buffer, pH 7.5, was pumped at a flow rate of 1 mL/min (pump P-50) and left to circulate through the disk for 2 h. Subsequently, the disk was rinsed with 50 mL of binding buffer. Any remaining free epoxy groups were blocked with 0.2 M Tris‚HCl buffer, pH 8.0. The disk was then rinsed with phosphate-buffered saline (PBS), pH 7.4, and stored at 4 °C until further use. The amount of immobilized GOX was controlled by in-process chromatography and by protein determination according to Lowry et al.27 RESULTS Fast Analyses with Compact Disks. Observations from earlier investigations show that an increase in the flow rates improves the performance of columns that are packed with perfusion support, of membranes, and of disks designed for membrane chromatography.5,25,28 By shortening the lengths of the columns and by using membranes or disks whose diameter (27) Lowry, O. H.; Rosenbrough, N. J.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275. (28) Josic, Dj.; Sˇtrancar, A. Proceedings of HPLC ‘95, Innsbruck, Austria, June 14-16, 1995; TH-L-69, p 39.
Figure 2. Separation of standard proteins obtained by QA anionexchange compact porous disk, 3 mm × 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, then 3 s isocratic 50% buffer B; flow rate, 10 mL/min; back pressure, 0.8 MPa; room temperature. Calibration proteins: 39 µg of Mb (peak 1), 130 µg of CA (peak 2), and 260 µg of STI (peak 3) in 20 µL of buffer A. Figure 1. (A, top) Construction of separation device containing a compact porous disk with 10 mm diameter and 3 mm layer thickness. Upper part: Construction elements of disk holder (details of element no. 8 in lower part of the figure). Lower part: Construction of the complete separation unit. Key: 1, 4, frits; 2, cartridge elementss polymeric tube; 3, compact, porous disk, 3 mm × 10 mm i.d.; 5, 11, 12, cartridge elements; 6, 10, O-rings; 7, 9, sample distributor; 8, disk holder. (B, bottom) Sample distribution, demonstrated by immobilization of injected ferritin on an epoxy-activated disk (cf. also ref 25).
is relatively large in comparison with the thickness of the layer, the question of sample distribution arises. It is of particular importance for membrane chromatography.5,25 In the case of membranes, satisfactory sample distribution can be achieved by using separation devices with large dead volumes in front of and behind the actual separation unit.5,25 However, performance of such a unit is impaired in the broadening of the peak (especially “tailing”) as a result of the large dead volume.21,25 The use of a sample distributor in membrane chromatography, as recommended by Josic et al.,25 improves the performance of the separation device with a diameter between 20 and 50 mm and reduces the dead volume. The market offers membrane units as cartridges whose construction can hardly be modified at all. However, it is possible to use a unit made of PS-DVB and GMA-EDMA, which are available in the form of loose plates. These plates can be installed in different cartridges. Figure 1A shows the improved construction for compact disks with a diameter smaller than 20 mm. Figure 1B shows that an almost perfect sample distribution was achieved with this unit. When compact porous disks were used, the size of the separation unit could be reduced to a diameter of 10 mm and layer thickness of 2-4 mm, without any reduction in separation performance (cf. Figures 2-4). Improved units of this kind reduce the period of time required for the separation of calibration
Figure 3. Cation-exchange chromatography of standard proteins on a compact disk. Chromatographic conditions: separation unit, a disk containing SO3 groups with a 10 mm diameter and 3 mm thickness; buffer A, 10 mM potassium phosphate, pH 5.5; buffer B, 1 M NaCl in buffer A; gradient time, 0-20% buffer B in 2 s, then in 1 s to 100% buffer B, and subsequently 1 s isocratic at 100% buffer B; flow rate, 10 mL/min; back pressure, 1.0 MPa; room temperature; detection, UV at 280 nm. Calibration proteins, 70 µg of RNA-se (peak 1), 300 µg of cytochrome c (peak 2), and 50 µg of lysozyme (peak 3) in 20 µL (volume of sample loop) of buffer A.
proteins to 20 s or less. This was the case with all separation modes that we investigated, including AE, CE, and HI modes (cf. Figures 2-4). The limiting factor was not the potential speed of Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
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Figure 4. Hydrophobic interaction chromatography of standard proteins on a compact porous disk. Chromatographic conditions: separation unit, a disk containing propyl groups with 10 mm diameter and 3 mm thickness; buffer A, 10 mM potassium phosphate buffer, containing 0.8 M (NH4)2SO4, pH 7.0; buffer B, 10% EtOH; gradient time, 0-100% buffer B in 2 s, then 5 s isocratic at 100% buffer B; flow rate, 10 mL/min; back pressure, 0.7 MPa; room temperature; detection, UV at 280 nm. Calibration proteins: 50 µg of Mb (peak 1), 100 µg of ovalbumin (OA) (peak 2), and 150 µg of STI (peak 3) in 20 µL (volume of sample loop) of buffer A.
the separation unit but the HPLC equipment and its software, which had not been constructed for such fast separations (cf. Materials and Methods and Figure 2). Despite this, all results were reproducible. This is demonstrated by nine subsequently performed separations of three calibration proteins under identical conditions as shown in Figure 2. Relative standard deviations of the peak areas were as follows: Mb, 1.4%; CA, 0.93%; and STI, 0.63%. Reproducibility of this kind is a prerequisite for the separation unit and equipment to be used for in-process control. Applications for In-Process Control During Isolation of Plasma Proteins. The power of the improved construction of the disk unit is demonstrated on the following examples. When R1-antitrypsin (AAT) is isolated from human plasma by preparative AE chromatography (AEC), transferrin (Tf) and human serum albumin (HSA) appear as main contaminants to be removed in the subsequent purification process.32 Figure 5 shows the chromatographic profile of a sample before the removal of the contaminating proteins (curve A). When the last purification step has been carried out, only the AAT peak is seen in the corresponding chromatogram (curve B). With this method, a statement about the results of the purification process can be made within 40 s. When clotting factor IX (FIX) is isolated from human plasma, contaminating proteins, chiefly HSA, Tf, and immunoglobulins, have to be removed in a first purification step carried out with (29) Tennikova, T.; Svec, F. J. Chromatogr. 1993, 646, 279-288. (30) Josic, Dj.; Baum, O.; Lo ¨ster, K.; Reutter, W.; Reusch, H. In Proceedings of Prep’92, Nancy, France Perrut, M., Ed.; May 1992; pp 113-117. (31) Josic, Dj.; Lim, Y.-P.; Sˇtrancar, A.; Reutter, W. J. Chromatogr. B 1994, 662, 217-226. (32) Josic, Dj.; Sˇ trancar, A.; Nur, I. FRG Patent P4407837.4, March 9, 1994.
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Figure 5. In-process analysis during R1-antitrypsin (AAT) production. (A) The sample contains AAT (5 mg/mL), human serum albumin (HSA, 5 mg/mL), and transferrin (Tf, 5 mg/mL) as impurities. (B) Purified AAT (5 mg/mL). Chromatographic conditions: separation unit, a DEAE-GMA-EDMA disk with 25 mm diameter and 3 mm thickness; buffer A, 10 mM Tris‚HCl, pH 7.4; buffer B, 1 M NaCl in buffer A: gradient time, 0-100% buffer B in 30 s; flow rate, 5 mL/ min, back pressure, 0.8 MPa; room temperature; detection, UV at 280 nm. Peak 1, Tf; peak 2, AAT; peak 3, HSA.
AEC. In a second purification step, heparin affinity chromatography, other vitamin-K-dependent coagulation factors, namely the factors II, VII, and X, are removed.6 Figure 6A shows the in-process sample, which contains contaminating plasma proteins. Figure 6B,C shows the profiles of two final products which are analyzed by fast AE chromatography. Both SDS-PAGE and determination of the specific activity of the FIX products (methods already described by Josic et al.6), along with in-process samples, have yielded the values that correspond to the profiles shown in Figure 6. The product whose profile is shown in Figure 6B was obtained through a combination of AEC and heparin affinity chromatography.6 The sample has a specific activity of about 140 IU FIX/ mg of protein. It contains FIX (peak 3) and one contaminant, with an apparent molecular mass of about 180 000 (peak 4). The FIX sample in Figure 6C was obtained by immunoaffinity chromatography with immobilized anti-FIX monoclonal antibodies.33 The sample has a specific activity of more than 200 IU/mg of protein and contains one contaminant, a polypeptide resulting from cleavage of FIX (peak 5). The answer to the status of FIX purification was given in less than 1 min. Figure 7 shows monitoring of the binding of GOX to an epoxyactivated disk as a model for in-process analysis of the immobilization of proteins to solid supports. At the beginning of the immobilization process, almost all the protein binds to the matrix. This leaves only a tiny amount of nonbound GOX in the flowthrough. The end of the immobilization process is reached when the peak in the flow-through is as high as the peak of the GOX solution in front of the unit. The differences between the peak areas of the GOX solutions before and after immobilization reveal the amount of immobilized protein. In the binding experiment shown in Figure 7, 1 mg of GOX was calculated to have bound to (33) Limentani, S. A.; Gowell, K. P.; Deitcher, S. R. Thromb. Haemost. 1995, 73 (4), 584-591.
Figure 6. In-process analysis, AE chromatography of samples taken from the production process of clotting factor IX (FIX). (A) Sample after anion-exchange chromatography, containing impurities (cf. ref 6). Chromatographic conditions: separation unit, a QA-GMA-EDMA disk with 10 mm diameter and 3 mm thickness; buffer A, 10 mM Tris‚ HCl, pH 7.4; buffer B, 1 M NaCl in buffer A; gradient time, 10% buffer B in 1 s, 10 s isocratic step with 10% buffer B, then in 1 s 30% buffer B, 8 s isocratic step with 30% buffer B, subsequently in 1 s 60% buffer B, followed by 7 s isocratic step with 60% buffer B, and finally in 1 s to 100% buffer B, and 11 s isocratic step with 100% buffer B; flow rate, 5 mL/min; back pressure, 0.5 MPa; detection, UV at 280 nm. Sample: 10 µg of Tf, 10 µg of IgG (peak 1), and 40 µg of HSA (peak 2) as impurities and 10 IU (about 40 µg) of FIX (peak 3) in 20 µL of buffer A. (B) For chromatographic conditions, see (A). Sample: FIX preparation Octanyne (Octapharma), containing 10 IU of FIX (peak 3) and some impurities with apparent molecular weights of about 180 000 in SDS-PAGE (peak 4) in 20 µL of buffer A. (C) For chromatographic conditions, see (A) Sample: FIX preparation (Mononine), purified by monoclonal antibodies, containing 10 IU of FIX (peak 3) and some smaller polypeptides (peak 5) in 20 µL of buffer A.
an epoxy-activated disk with a 10 mm diameter and 3 mm layer thickness. The result agreed well with the difference in protein concentration, which was found by determining the protein by the Lowry method27 in the samples before and after immobilization. It has been shown before that enzymes, when immobilized to such compact porous disks, are able to cause fast conversion of substrate in flow-through.23 The disk with immobilized enzyme GOX is used for measuring the glucose concentration during monitoring of different types of fermentation processes by means of flow injection analysis system (FIA). The disk is used instead of a column with immobilized GOX.34,35 DISCUSSION In the course of the last 10 years, the separation time of HPLC analyses of biopolymers, initially in the range of 30-90 min or more, has been reduced considerably. The development of (34) Nielsen, J.; Nikolajsen, K.; Villadsen, J. Biotechnol. Bioeng. 1989, 33, 11271134. (35) Garn, M.; Gisin, M.; Thommen, Ch.; Cevey, P. Biotechnol. Bioeng. 1989, 34, 423-428.
Figure 7. Monitoring of the binding of glucose oxidase (GOX) during immobilization to an epoxy-activated disk, 3 mm × 10 mm In-process control was carried out on a QA anion-exchange compact porous disk, 3 mm × 10 mm i.d. 1 mg of GOX bound on the disk. Chromatographic conditions: buffer A, 10 mM Tris‚HCl, pH 7.4; buffer B, buffer A + 1 M NaCl; gradient time, 0-100% buffer B in 35 s; flow rate, 3 mL/min; back pressure, 0.4 MPa; volume of sample loop, 100 µL; detection, UV at 280 nm. (A) Chromatogram at the beginning of the immobilization. Protein concentration, 0.6 mg/mL; volume, 5 mL. (B) Chromatogram at the end of the immobilization. Protein concentration, 0.3 mg/mL; volume, 6.8 mL (dilution due to dead volume in the pump, tubing, and cartridge).
polymeric or silica gel supports based on spheric particles with diameters less than 5 µm and large pores played a decisive role in enhancing separation performance and reducing separation time. Another contributing factor was the introduction of nonporous supports with particle sizes between 1 and 10 µm.1 As far as the use of porous supports for protein separation is concerned, a simple time-oriented optimization of the process can be achieved. This was confirmed by the investigations carried out by Kopaczewicz et al.19 A similar result for Mono Q support can be achieved (not shown here). The application of corresponding gradients and flow rates allows a reduction of analysis time in the field of protein separation, even if regular analytical columns (usually 50 mm × 5 mm i.d., up to 150 mm column length) are used for this purpose. Columns packed with nonporous (micropellicular) supports with particle sizes smaller than 5 µm show an outstanding performance, separating biopolymers within a few minutes or even in a few seconds (cf. refs 1, 9-12). However, when columns packed with nonporous, microparticle-sized supports are used, high performance and short separation times are achieved at the cost of high back pressure, and of rather limited capacity as well. Higher temperatures, as proposed by Csen and Horvath,1 even when combined with rather short columns with micropellicular supports, have so far been applied only to the separation of proteins in reversed-phase mode. It therefore requires intensive sample preparation, even when the systems are used for in-process analyses. This, in turn, does not agree with the requirement of a simple and robust unit for this kind of application. Despite promising results in the analytical laboratory the use of microAnalytical Chemistry, Vol. 68, No. 19, October 1, 1996
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pellicular supports at high temperatures for in-process analyses can be considered in single cases only, at least for the time being. Recently developed, highly porous bulk materials have brought a significant improvement in the field of protein separation. The supports used for perfusion chromatography as well as the newly developed source supports (Pharmacia) are suited to make fast analyses of biopolymers within a very short time. The application of specially developed chromatographic instruments and software allows perfusion materials to separate biopolymers in less than 1 min.2 Columns with perfusion materials have been used successfully several times for in-process analysis of production and purification of proteins.2,36,37 When columns packed with perfusion or source supports are used, the problems of sampling and sample pretreatment have to be solved in each single case. The main problem, the transportation of solid or colloidal particles on to the column, which can cause its plugging, has to be prevented by using adequate filters or other technical means. Air bubbles should be removed as well. Compared to chromatographic colums packed with porous supports, the separation of biopolymers can also be accelerated by using the method of HPMC. The main problem with the use of HPMC units, both membranes and compact disks, is their lack of stability at higher pressures and their dead volumes. The specific way in which samples are distributed on compact disks ensures high reproducibility of all performed analyses.25 Besides, the separation unit is protected against destruction. However, this sample distributor is responsible for a dead volume which is still too big when the diameter of the cartridge is smaller than 20 mm. Because of these dead volumes and the fact that a risk of destruction still remains at high flow rates, reproducible analyses cannot be carried out in less than 30 s with separation units that have diameters of more than 20 mm. The construction of the unit as illustrated in Figure 1 with a diameter of 10 mm guarantees a favorable sample distribution at low back pressure and provides protection against its destruction. This construction is a milestone for fast analyses within seconds (Figures 2-7). The alternative proposed by Belenkii and Malt’sev,24 Tennikova and Svec,29 Reif and Freytag,5 and Josic et al.,30,31 which is based on the application (36) Nadler, T. K.; Paliwal, S. K.; Regnier, F. E. J. Chromatogr. A 1994, 659, 317-320. (37) Hunt, A. J.; Lynch, P. D.; Londo, T.; Dimond, P.; Gordon, N. F.; McCormack, T.; Schultz, A.; Percoskic, M.; Cao, X.; Mc Gratz, J. P.; Putney, S.; Hamilton, R. A. J. Chromatogr. A 1995, 708, 61-70. (38) Josic, Dj.; Sˇ trancar, A; Koselj, P.; Podgornik, A. Eur. Pat. Appl. PCT/EP95/ 03333, August 22, 1995.
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of step gradients to speed up analyses, is useful only to a limited degree. In our earlier experiments, five calibration proteins were separated on baseline within 5 min by HIC disk with a 25 mm diameter.30,31 The minimal separation time achieved using such separation units under the chosen conditions ranged from approximately 45 s to 1 min per separated component and gradient step. A further reduction of gradient time resulted in an incomplete elution of some components, which appear as an additional peak in the next step.31 The in-process control both of fermentation processes and of downstream processing operations has become increasingly important and necessary in biotechnology. The on-line removal of solid and colloidal particles, agglomerates, and cells has two main objectives, reliable results and a longer life of the applied analytical unit.7,8 The disks used here and the hardware can provide measures for the prevention of such problems. As binding of the sample takes place on the surface of the unit (Sˇ trancar, A.; Josic, Dj., submitted for publication), the application of the sample can be carried out by a cross-flow mode, replacing the flow-through method which is necessary in the case of columns. The support is chemically stable, allowing its sanitation with acidic and alkaline solutions.22 The mechanical stability of the support, that is, its compact structure, makes it possible to remove deposited solid particles from its surface by ultrasonication.38 CONCLUSIONS Separation units containing compact disks can be used for fast separations of biopolymers within a few minutes. If the construction of the cartridge, which contains a compact porous disk as a separation unit, is improved along specific lines, separations can be carried out within a few seconds. When these units are used in the analytical field, the time limit is set not by the speed of the separation itself but by the characteristics of the hardware, above all the dead volume. Through adequate construction of the hardware, the separation units with compact porous disks can be used for fast analyses of fermentation processes and downstream processing. Received for review March 25, 1996. Accepted July 15, 1996.X AC960292F X
Abstract published in Advance ACS Abstracts, August 15, 1996.
