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Cation-exchange displacement chromatography for the purification of recombinant protein therapeutics from variants
Journal of Biotechnology 66 (1998) 125 – 136
Cation-exchange displacement chromatography for the purification of recombinant protein therapeutics from variants Kristopher A. Barnthouse a, William Trompeter b, Rodrick Jones b, Prasad Inampudi b, Randall Rupp b, Steven M. Cramer a,* a
Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA b Regeneron Pharmaceutical Inc., Rensselaer, NY 12144, USA
Received 20 January 1998; received in revised form 3 August 1998; accepted 10 August 1998
Abstract Removal of low level impurities that are closely related to the bioproduct is a commonly encountered challenge in the purification of biopharmaceuticals. These separations are typically carried out by using shallow linear salt gradients at relatively low column loadings, significantly limiting the throughput of the purification process. In this manuscript we examine the utility of displacement chromatography for the purification of recombinant human brain-derived neurotrophic factor, rHuBDNF. The utility of displacement chromatography is compared to gradient elution for the removal of variants of the rHuBDNF. The results demonstrate that displacement chromatography is capable of achieving high yields and purity at high column loadings. Displacements developed on 20 mm and 50 mm cation-exchange resins are shown to provide 8-fold and 4.5-fold increases in production rates, respectively, when compared to an existing linear gradient elution operation. These results demonstrate the efficacy of displacement chromatography for the purification of therapeutic proteins from complex feed streams. © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Displacement chromatography; Protein purification; Complex mixture; rHuBDNF; Variant; Protamine sulfate; Productivity; Comparison
1. Introduction Preparative ion-exchange chromatography has been widely utilized in step and continuous gradi* Corresponing author. Tel: + 1 518 2766198; e-mail: [email protected]
ent modes by the biotechnology industry for the purification of protein therapeutics (Yamamoto et al., 1987; Scopes, 1996). Typically, these modes of chromatography employ increasing salt concentrations for elution. Unfortunately salt elution has the concomitant effect of reducing the effective separation factor between products and impuri-
0168-1656/98/$ - see front matter © 1998 Published by Elsevier Science B.V. All rights reserved. PII S0168-1656(98)00131-X
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ties. For this reason, step and continuous gradient chromatography often require relatively large separation factors for high-throughput preparative separations. An attractive alternative for overcoming throughput limitations of preparative gradient chromatography is operation of the column in displacement mode. Displacement chromatography is performed in a manner operationally similar to step gradient chromatography in that the column is subjected to sequential changes in inlet conditions (Cramer, 1992). The column is initially equilibrated with a carrier of low ionic strength, after which a feed pulse is introduced under conditions of high retention, followed by a constant infusion of a displacer solution into the column. The displacer is chosen such that it has a higher dynamic affinity for the stationary phase than any of the feed components and thus competes favorably with the feed solutes for adsorption sites on the stationary phase. Under appropriate conditions the displacer induces the feed components to develop into adjacent ‘square wave’ zones of concentrated and purified material, all of which exit the column ahead of the displacer front. Following the breakthrough of the displacer, the column can be regenerated using a buffer of high ionic strength, commonly accompanied by an extreme in pH. The column can then be re-equilibrated with the initial carrier buffer. A general, practical discussion of displacement chromatography is provided by Cramer (1992). Several investigators have studied ion-exchange displacement chromatography of proteins using model systems. Horvath and co-workers have employed chondroitin sulfate to displace b-galactosidase (Liao and Horvath, 1990) and b-lactoglobulins (Liao et al., 1987; Lee et al., 1988). Jen and Pinto have employed polyvinyl sulfonic acid (Jen and Pinto, 1990) and dextran sulfate (Jen and Pinto, 1991) to separate a mixture of moderately retained proteins. Cramer and coworkers (Gerstner and Cramer, 1992a,b; Gadam et al., 1993; Jayaraman et al., 1993; Gadam and Cramer, 1994; Brooks and Cramer, 1996) have identified a variety of efficient polyelectrolyte displacers for protein purification including DEAE-
dextran, dextran sulfate, protamine, heparin, and pentosan polysulfate. Some research has also been carried out using displacement chromatography for the purification of proteins from a variety of natural biological mixtures (Torres et al., 1985; Dunn et al., 1988; Torres and Peterson, 1990; Ghose and Mattiason, 1991; Vogt and Freitag, 1997). An important recent advance has been the discovery that low molecular mass (B 2000 Da) displacers can be successfully employed for protein purification in ion-exchange displacement systems. These include dendrimers (Jayaraman et al., 1995), protected amino acids (Kundu et al., 1995a), antibiotics (Kundu et al., 1995b), and relatively nontoxic functionalized sucrose (Kundu et al., 1997). Despite the attractive advantages of displacement chromatography, there are no reports in the literature to date on the purification of recombinant protein therapeutics by displacement chromatography. In this manuscript we examine the utility of displacement chromatography for the purification of recombinant human brainderived neurotrophic factor, rHuBDNF. The rHuBDNF employed in this study was provided by Regeneron Pharmaceutical Inc. (Tarrytown, NY and Rensselaer, NY) as a partially purified mixture, pooled from an upstream chromatographic step. The protein is a dimer with an approximate molecular mass of 27 kDa. This growth factor is known to be quite basic, with a pI greater than 10. The rHuBDNF is expressed in E. coli and is harvested as an inclusion body. The purification of rHuBDNF by displacement chromatography is particularly challenging since rHuBDNF is very strongly bound to the cationexchange material. Furthermore, there are several variants of rHuBDNF which are extremely difficult to remove from the bioproduct of interest.
2. Materials and methods
2.1. Materials Regeneron Pharmaceutical Inc. provided the rHuBDNF feed stock used for the displacement
K.A. Barnthouse et al. / Journal of Biotechnology 66 (1998) 125–136
experiments. Two different feed lots were employed. A feed stock of 0.4 mg ml − 1 protein concentration at 87% purity was utilized for the HS/M displacements at loadings per column volume of 2.1 and 11.0 mg ml − 1. All other experiments utilized a feed stock containing 1.25 mg ml − 1 protein at 87.5% purity (note: this is the same feed stock that was employed for the linear gradient process). Each feed stock was buffered with 50 mM sodium phosphate, pH 7.0, containing 275 mM NaCl. Protamine Sulfate Grade X from salmon (P4020, Lot 21H0519) was purchased from Sigma Chemical (St. Louis, MO, USA). Sodium Phosphate, phosphoric acid, acetonitrile, and TFA were purchased from Fisher Scientific (Pittsburgh, PA, USA). A POROS HS/M (4.6 ×100 mm) column used for initial displacer screening and bulk POROS HS/M resin were donated by PerSeptive Biosystems (Framingham, MA, USA). Displacement experiments were carried out using both 50 and 75 cm column lengths which were obtained by arranging 2 and 3 columns in series, respectively. A Vydac C4 (4.6× 250 mm) column was purchased from The Separations Group (Hisperia, CA, USA). OmegaChrom columns (0.46× 250 mm) were purchased from Upchurch Scientific (Oak Harbor, WA, USA).
2.2. Apparatus All displacement experiments were carried out using a Waters 625 LC system including a Waters 996 photodiode array detector, and a Waters Fraction Collector. Data collection and analysis was performed using Waters Millennium Chromatography Manager. Analysis of the collected fractions was carried out using a Waters 600S Gradient Controller, a Waters 626 Solvent Delivery Unit, a Waters PDA 996 photodiode array detector and a Waters 717 WISP autosampler with cooling module. A Waters Temperature Control Module was utilized for high-temperature reversed-phase analysis. Column packing was performed using PerSeptive Biosystems Self-Pack column packing apparatus.
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2.3. Analytical high-temperature RPLC Displacement fractions were analyzed using an acetonitrile gradient with a constant concentration of TFA as an ion-pairing agent. All analyses were performed using a Vydac C4 (4.6×250 mm) column at 65°C. Injection volume was adjusted to deliver 50 mg of total protein. The effluent was monitored at 215 nm.
2.4. Analytical ion-exchange chromatography The column feed stock was analyzed by ion-exchange chromatography using a 4.6× 250-mm columns packed with POROS HS50 resin. A gradient of sodium chloride was performed from 200 mM to 1200 mM over 10 column dead volumes. The mobile phase was buffered with 50 mM sodium phosphate, pH 7.0.
2.5. Displacement chromatography In all displacement experiments, the columns were initially equilibrated with the carrier and then sequentially perfused with feed, displacer and regenerant solutions. For all displacements, column loading was performed at a superficial mobile phase velocity of 180 cm h − 1 (0.50 ml min − 1) and the displacer was perfused at 90 cm h − 1 (0.25 ml min − 1). The mobile phase carrier was 50 mM sodium phosphate, 500 mM sodium chloride, pH 7.0. Displacer concentrations of 3 mM and 0.5 mM protamine sulfate were employed. Column lengths were 50 or 75 cm. The column effluent was monitored at 280 nm. Fraction sizes were 500 ml (for experiments using 3.0 mM protamine) and 1000 ml (for experiments using 0.5 mM protamine).
2.6. Column packing Two columns (4.6× 250 mm) of POROS HS/ M, three columns of (4.6×250 mm) of POROS HS50, and one (4.6× 50 mm) column of POROS HS50 were packed according to the following: Dry resin was slurried with packing buffer (50 mM sodium phosphate+ 150 mM sodium chloride) at a concentration of 0.1 g ml − 1. Packing
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was performed at constant velocity of 7200 cm h − 1 using the PerSeptive Self-Pack apparatus. Packing was complete after 150 column volumes of packing buffer were passed. After packing, each column was tested for asymmetry and plate count using a 20-ml pulse of 100 mM sodium nitrate as an unretained tracer with a mobile phase of 500 mM sodium chloride buffered at pH 7.0.
was first identified and used as a displacer by Gerstner (Gerstner and Cramer, 1992a). Protamine sulfate (5 kDa) is rich in arginine residues and possesses a high affinity for cation-exchange resins. Protamine sulfate itself has a therapeutic application as an antidote to the anticoagulant heparin (The United States Pharmacopeia, 1990).
3. Results and discussion
Initial displacement experiments were performed on an analytical (4.6× 500 mm) column packed with (20 mm) POROS HS/M resin. Fig. 2 presents a histogram of a displacement performed using 3 mM protamine displacer at a loading per column volume of 20.0 mg ml − 1, eight-times higher than used for the gradient process. As seen in the figure, protamine was able to produce an efficient displacement of the strongly bound rHuBDNF. Fractions from the column effluent were analyzed by RPLC and the resulting data were used to generate Fig. 3a-c. Although completely separated pure zones of product and impurities were not achieved, the impurities and product are found in highly enriched zones. This can be attributed to the difficulty of the separation. Difficult separations approach their final constant-pattern displacement profiles very slowly and may require long columns of impractical length (Antia and Horvath, 1991). Also, the finite degree of overlap that exists in constant-pattern displacement profiles (shock layer thickness) is also expected to be greater for very close separations (Zhu and Guiochon, 1994). Nevertheless, as seen in Fig. 3, the displacement process resulted in significant purification of the rHuBDNF with some fractions reaching as high as 96% purity. The product pool of all fractions greater than 85% purity (fractions 9 through 30) resulted in a 72.9% yield of product with a purity of 93.6% (note: this compares favorably to gradient chromatography which produced a yield of 67.9% at 93.1% purity at 8 times lower loadings). In addition to producing high yields and purity, the displacement process also resulted in an 8-fold concentration of the rHuBDNF to ap-
An analytical high temperature reversed-phase chromatogram of the rHuBDNF feed stream is shown in Fig. 1a. As seen in the figure, the feed consists of the rHuBDNF main peak along with several variants. The objective of this ion-exchange step was to reduce the variants below acceptable limits. Several ion-exchange resins were screened for their selectivity for this separation. The POROS HS resin (PerSeptive Biosystems, Framingham, MA) was found to have the highest selectivity for this separation and was observed to reproducibly reduce the variants to an acceptable level by linear gradient chromatography. However, in order to accomplish this separation, the linear gradient process was limited to low column loadings and very shallow linear gradients. Also seen in Fig. 1a are several later eluting impurity peaks that have been identified as host cell proteins. Removal of the host cell proteins is accomplished further downstream and is not the primary focus of the ion-exchange purification step. An analytical scale ion-exchange linear salt gradient was performed and the resulting chromatogram (Fig. 1b) reveals two difficulties encountered with this separation. First, the lack of baseline resolution or shoulders indicates that the protein variants have chromatographic properties very close to that of the bioproduct. Second, the rHuBDNF elutes at a very high mobile phase salt concentration (ca. 900 mM sodium chloride). This indicates that both the product and variants are strongly binding and implies the use of a very high affinity displacer. The displacer chosen for this work, protamine sulfate from salmon,
3.1. Displacements on POROS HS/M (20 mm) particles
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Fig. 1. Analytical chromatography of feed stock. (a) High-temperature reversed-phase acetonitrile gradient; Sample: 50 mg injection of rhuGFP ion-exchange feed; Column: 4.6 × 250 mm Vydac C4; (b) Analytical chromatogram on ion-exchange, pH 7.0; Sample: 20 mg of rhGFP ion-exchange feed; Column: 4.6 × 250 mm Poros HS50 strong cation-exchanger.
proximately 10 mg ml − 1. Thus, this displacement process resulted in simultaneous concentration and purification which is a well-established benefit of displacement mode chromatography (Horvath
and Brunner, 1985). Dynamic binding capacity data indicated that this displacement experiment was carried out at 33.6% of the total column capacity for this feed stock.
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Fig. 2. Histogram of ion-exchange displacement: 20 mg ml − 1 loading. Histogram showing total displaced protein (product and variants) and protamine sulfate displacer. Constructed from high-temperature reversed-phase analysis of displacement fractions.
Improved performance at high column loading is counterintuitive for practitioners of elution chromatography. In contrast to elution chromatography in which increased loading results in an increase in peak widths and hence overlap, increased loading in displacement chromatography merely increases the width of purified zones. Since in a developed displacement train the degree of overlap between zones is constant with respect to loading, increased loading can often improve pool purity, product yields and productivity. Results from two displacement experiments carried out at lower column loading illustrate this effect. A displacement experiment with a mass loading per column volume of 2.1 mg ml − 1 (similar to the loading of the linear gradient operation), resulted in a purity of 90.8% with a 62.5% yield. This purification lacked sufficient mass to achieve good recovery. By increasing the column loading to 11.0 mg ml − 1 the product purity and yield were increased to 92.8% and 72.6%, respectively. As described above, at an even higher loading of 20.0 mg ml − 1, the displacement con-
tinued to perform well, with concomitant increases in productivity. Analytical RPLC chromatograms of representative fractions found early, intermediate, and late in the 20 mg ml − 1 loading displacement experiment are presented in Fig. 4. While early displacement fractions are enriched in the variants found before the main peak, later fractions are enriched in variants found after the main product peak. These results indicate that the ion-exchange resin and the reversed-phase material appear to have a similar selectivity trend for the variants. This is unusual in that one would expect the selectivity of these two classes of resins to be quite different. Thus, these results indicate that the selectivity of the POROS ion-exchanger may be aided by nonspecific, hydrophobic interactions between the variants and the stationary phase material.
3.2. Displacements on POROS HS50 (50 mm) particles Purifications at bench-scale are often best performed using columns packed with small particle
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Fig. 3. Displacement fraction purity: 20 mg ml − 1 loading. Composition of fractions as determined by analytical high-temperature reversed-phase. Reported are rhuGFP main peak and variants as percent mass. (a) Feed and fractions 2 – 12; (b) Fractions 13 – 24; (c) Fractions 25 – 34.
resins due to their high efficiency and high capacity. In contrast, for manufacturing-scale chromatography, the performance benefits of smaller particle diameter resins are often outweighed by the drawbacks of higher pressure drop and resin cost. Since costly high pressure columns and
equipment would be required for performing process-scale chromatography with the POROS HS/ M resin, the utility of POROS HS50 (50 mm) resin for displacement chromatography was evaluated. A series of displacement experiments were carried out on the 50 mm material with a displacer
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Fig. 4. High temperature reversed-phase assay of three displacement fractions: 20 mg ml − 1 loading. Overlay of fractions collected from points early (fraction 16), intermediate (fraction 18), and late (fraction 31) of the displacement train. Note the variant enrichment in the early and late fractions.
concentration of 3.0 mM. These experiments did not result in acceptable reductions in the variants due to mass transport limitation inherent in the 50 mm material. Even when these experiments were carried out using longer column lengths (75 cm) the product pools were still below purity specifications. Overcoming the mass transport limitations of the 50 mm particles necessitated a six fold reduction in displacer concentration (to 0.5 mM) from that used with the 20 mm materials. A displacement experiment was carried out at a loading per column volume of 11.0 mg ml − 1 and at a displacer concentration of 0.5 mM. Fractions from the column effluent were analyzed by RPLC and
the fraction purity is presented in Fig. 5. As seen in the figure, the displacement process resulted in significant purification of the rHuBDNF with some fractions reaching as high as 95.4% purity. Pooling all fractions of greater than 85% purity (fractions 10 through 31) resulted in a 77.9% yield of product with a purity of 93.4%. Thus, by reducing the displacer concentration to 0.5 mM the results with the 50 mm materials became comparable to the best result with the 20 mm resin. Reducing the displacer concentration has several benefits. First, it is well established that reducing the displacer concentration lowers the concentrations of the product and impurity zones. These wider, less concentrated product zones can
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Fig. 5. Displacement fraction purity: 0.5 mM displacer concentration. Composition of fractions as determined by analytical high-temperature reversed-phase. Reported are rhGFP main peak and variant c 4 as percent mass. (a) Feed and fractions 3 – 13; (b) Fractions 14 – 25, (c) Fractions 26–35.
improve product yield due to a decrease in the relative amount of overlapping of enriched product and impurity zones, thereby diminishing the effects of mass transport limitations. Further, if desorption kinetics play a limiting role, slowing down the separation by reducing the displacer concentration may allow local equilibrium to be achieved.
3.3. Impacts on chromatographic producti6ity The production rate of rHuBDNF for the ionexchange step can be defined (Eq. (1)) in terms of the column loading of the rHuBDNF and its variants (mass per column volume), the yield of the step (Y), the purity of rHuBDNF in the feed
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Table 1 Cycle times computed for gradient and displacement purificationsa Mode/particle size
tequilibration (h)
tloading (h)
tseparation (h)
tregeneration (h)
tcycle (h)
Displacement/20 mm Displacement/50 mm Gradient/50 mm
4.0 4.0 4.0
16.0 8.8 1.9
4.5 13.2 11.0
22.1 22.1 23.6
46.7 48.2 40.5
a
Feed concentration and all volumetric flow rates (number of column volumes per hour) are equivalent for each purification.
(Pfeed), and the cycle time of the run (tcycle) (note: product purity and yield are defined with respect to the rHuBDNF and its variants; the small amount of host cell proteins were not included in these calculations) (Gallant et al., 1995, 1996). PR =
mfeed Vcolumn
YPfeed tcycle
(1)
Maximizing the production rate is an often sought after goal, as this allows the generation of the greatest amount of product in the least amount of time while using the least amount of stationary phase material. Increasing the production rate of a bottleneck purification step can impact the productivity of the entire purification process. The cycle time in Eq. (1) is the amount of time required for one complete cycle of the chromatographic step. This includes the time required for equilibration, loading, separation, and column regeneration. tcycle = tequilibration + tfeed +tseparation +tregeneration (2) The column regeneration time employed by the manufacturer for this process includes the time required for clean-in-place and sanitization procedures in addition to regeneration. Although it is more common to perform clean-in-place procedures after several cycles and to perform sanitization only between batches (Sofer and Nystrom, 1991), the current linear gradient process employs these aggressive measures to remove very strongly binding impurities and contaminants after each chromatographic use. Table 1 contains the various contributions to the cycle time obtained from the 20 mm displacement, the 50 mm displacement, and the linear gradient process for the same feed
lot. As seen in the table, while the equilibration and regeneration times remain relatively similar for these processes, the loading and separation times are quite different. The productivity of these separations is presented in Table 2. Both displacement processes resulted in significant improvements in the production rate. The 20 mm and 50 mm displacements resulted in an eight fold and 4.5 fold improvement, respectively, as compared to the current gradient process. It is worth noting that the production rate would be significantly higher if the inordinately long regeneration time were not employed. The 50 mm displacement provides a significant increase in production rate while utilizing existing manufacturing equipment. Without further optimization or equipment changes, the current practice of processing the ion-exchange feed in four separate sub-lots could be discontinued, thereby greatly impacting the productivity of the entire process. Other possible benefits include increased resin lifetime due to reduced exposure to harsh regenerating conditions and savings in labor costs. The increase in productivity of a 20-mm displacement can provide even greater savings of manufacturing time and resin volume but would require the added expense of high-pressure columns and equipment. A detailed economic analysis of the process is currently being performed to determine the practicality of using 20 mm resin in displacement mode. The cycle time of each displacement was strongly influenced by the loading time while the cycle time of the linear gradient was not (Table 1). Thus, an increase in the feed concentration brought about by improvements in upstream processes would result in further improvements to the
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Table 2 Productivity comparison of displacement and gradient elution purifications Mode/particle size
Loading (g l−1)
Feed purity (%) tcycle (h)
Yield (%)
Productivity (mg l−1 h−1)
Improvement w/r/t gradient
Displacement/20 mm Displacement/50 mm Gradient/50 mm
20.0
88.2
46.7
73.8
278.7
8.0x
11.0
87.1
48.2
77.9
155.0
4.5x
87.4
40.5
67.9
34.6
N/A
2.36
productivity of the displacement processes relative to the linear gradient process. The results presented in this paper demonstrate that displacement chromatography is capable of purifying rHuBDNF from its variants with both high yields and purity at elevated column loadings. Further, the displacement process provides a significant improvement in the productivity of this ion-exchange operation as compared to the linear gradient process.
Acknowledgements This research was supported by Grant No. GM47372 from the National Institutes of Health. The authors also wish to thank Dr Robert MacColl and Leslie Eisele of the Biochemistry Core at the Wadsworth Center, New York State Department of Health, for the use of their equipment and facilities.
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Gadam, S., Cramer, S.M., 1994. Salt effects in anion-exchange displacement chromatography – comparison of pentosan polysulfate and dextran sulfate displacers. Chromatographia 39 (7/8), 409 – 418. Gadam, S., Jayaraman, G., Cramer, S.M., 1993. Characterization of high affinity dextran based displacers. J. Chromatogr. 630, 37 – 52. Gallant, S.R., Kundu, A., Cramer, S.M., 1995. Optimization of step gradient separations: consideration of nonlinear adsorption. Biotechnology & Bioengineering. 47, 355 – 372. Gallant, S.R., Vunnum, S., Cramer, S.M., 1996. Optimization of preparative ion-exchange chromatography of proteins: linear gradient separations. J. Chromatogr. A. 725, 295 – 314. Gerstner, J.A., Cramer, S.M., 1992a. Cation-exchange displacement chromatography of proteins with protamine displacers: effects of induced salt gradients. Biotech. Prog. 8, 540 – 545. Gerstner, J.A., Cramer, S.M., 1992b. Heparin as a non-toxic displacer for anion-exchange displacement chromatography of proteins. BioPharmacology 5 (9), 42 – 45. Ghose, S., Mattiason, B.J., 1991. Evaluation of displacement chromatography for the recovery of lactate dehydrogenase from beef heart under scale-up conditions. J. Chromatogr. 547, 145 – 153. Horvath, Cs., Brunner, F. (Eds.), 1985. The Science of Chromatography, Elsevier, Amsterdam, p. 185. Jayaraman, G., Gadam, S., Cramer, S.M., 1993. High affinity dextran based displacers for ion-exchange displacement chromatography of proteins. J. Chromatogr. 630, 53 – 68. Jayaraman, G., Li, Y., Moore, J.A., Cramer, S.M., 1995. Ion-exchange displacement chromatography of proteins: dendritic polymers as novel displacers. J. Chromatogr. 702, 143 – 155. Jen, S.C., Pinto, N., 1990. Use of the sodium salt of poly(vinylsulfonic acid) as a low molecular weight displacer for protein separations by ion-exchange displacement chromatography. J. Chromatogr. 519, 87 – 98. Jen, S.C., Pinto, N., 1991. Dextran sulfate as a displacer for the displacement chromatography of pharmaceutical proteins. J. Chromatogr. Sci. 29, 478 – 484. Kundu, A., Shukla, A., Barnthouse, K.A., Moore, J., Cramer, S.M., 1997. Displacement chromatography of proteins using sucrose octasulfate. BioPharmacology 10 (5), 64 – 68.
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Kundu, A., Vunnum, S., Jayaraman, G., Cramer, S.M., 1995a. Protected amino acids as novel low molecular mass displacers in ion-exchange displacement chromatography. Biotech. & Bioeng. 48, 452–460. Kundu, A., Vunnum, S., Cramer, S.M., 1995b. Antibiotics as low molecular weight displacers in ion-exchange displacement chromatography. J. Chromatogr. 707, 57–67. Lee, A.L., Liao, A.W., Horvath, Cs., 1988. Tandem separation schemes for preparative high-performance liquid chromatography of proteins. J. Chromatogr. 443, 182– 191. Liao, A.W., Horvath, Cs., 1990. Purification of b-galactosidase by combined frontal and displacement chromatography. Annals N.Y. Acad. Sci. 589, 182–191. Liao, A.W., Rassi, Z.L., LeMaster, D.M., Horvath, Cs., 1987. High-performance displacement chromatography of proteins: separation of b-lactoglobulins A and B. Chromatographia 24, 881 –885. Scopes, R.K., 1996. Protein purification in the nineties. Biotechnol. Appl. Biochem. 23, 197–204. Sofer, G.K., Nystrom, L.-E. (Eds.), 1991. Process Chromatog-
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Cation-exchange displacement chromatography for the purification of recombinant protein therapeutics from variants Kristopher A. Barnthouse a, William Trompeter b, Rodrick Jones b, Prasad Inampudi b, Randall Rupp b, Steven M. Cramer a,* a
Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA b Regeneron Pharmaceutical Inc., Rensselaer, NY 12144, USA
Received 20 January 1998; received in revised form 3 August 1998; accepted 10 August 1998
Abstract Removal of low level impurities that are closely related to the bioproduct is a commonly encountered challenge in the purification of biopharmaceuticals. These separations are typically carried out by using shallow linear salt gradients at relatively low column loadings, significantly limiting the throughput of the purification process. In this manuscript we examine the utility of displacement chromatography for the purification of recombinant human brain-derived neurotrophic factor, rHuBDNF. The utility of displacement chromatography is compared to gradient elution for the removal of variants of the rHuBDNF. The results demonstrate that displacement chromatography is capable of achieving high yields and purity at high column loadings. Displacements developed on 20 mm and 50 mm cation-exchange resins are shown to provide 8-fold and 4.5-fold increases in production rates, respectively, when compared to an existing linear gradient elution operation. These results demonstrate the efficacy of displacement chromatography for the purification of therapeutic proteins from complex feed streams. © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Displacement chromatography; Protein purification; Complex mixture; rHuBDNF; Variant; Protamine sulfate; Productivity; Comparison
1. Introduction Preparative ion-exchange chromatography has been widely utilized in step and continuous gradi* Corresponing author. Tel: + 1 518 2766198; e-mail: [email protected]
ent modes by the biotechnology industry for the purification of protein therapeutics (Yamamoto et al., 1987; Scopes, 1996). Typically, these modes of chromatography employ increasing salt concentrations for elution. Unfortunately salt elution has the concomitant effect of reducing the effective separation factor between products and impuri-
0168-1656/98/$ - see front matter © 1998 Published by Elsevier Science B.V. All rights reserved. PII S0168-1656(98)00131-X
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ties. For this reason, step and continuous gradient chromatography often require relatively large separation factors for high-throughput preparative separations. An attractive alternative for overcoming throughput limitations of preparative gradient chromatography is operation of the column in displacement mode. Displacement chromatography is performed in a manner operationally similar to step gradient chromatography in that the column is subjected to sequential changes in inlet conditions (Cramer, 1992). The column is initially equilibrated with a carrier of low ionic strength, after which a feed pulse is introduced under conditions of high retention, followed by a constant infusion of a displacer solution into the column. The displacer is chosen such that it has a higher dynamic affinity for the stationary phase than any of the feed components and thus competes favorably with the feed solutes for adsorption sites on the stationary phase. Under appropriate conditions the displacer induces the feed components to develop into adjacent ‘square wave’ zones of concentrated and purified material, all of which exit the column ahead of the displacer front. Following the breakthrough of the displacer, the column can be regenerated using a buffer of high ionic strength, commonly accompanied by an extreme in pH. The column can then be re-equilibrated with the initial carrier buffer. A general, practical discussion of displacement chromatography is provided by Cramer (1992). Several investigators have studied ion-exchange displacement chromatography of proteins using model systems. Horvath and co-workers have employed chondroitin sulfate to displace b-galactosidase (Liao and Horvath, 1990) and b-lactoglobulins (Liao et al., 1987; Lee et al., 1988). Jen and Pinto have employed polyvinyl sulfonic acid (Jen and Pinto, 1990) and dextran sulfate (Jen and Pinto, 1991) to separate a mixture of moderately retained proteins. Cramer and coworkers (Gerstner and Cramer, 1992a,b; Gadam et al., 1993; Jayaraman et al., 1993; Gadam and Cramer, 1994; Brooks and Cramer, 1996) have identified a variety of efficient polyelectrolyte displacers for protein purification including DEAE-
dextran, dextran sulfate, protamine, heparin, and pentosan polysulfate. Some research has also been carried out using displacement chromatography for the purification of proteins from a variety of natural biological mixtures (Torres et al., 1985; Dunn et al., 1988; Torres and Peterson, 1990; Ghose and Mattiason, 1991; Vogt and Freitag, 1997). An important recent advance has been the discovery that low molecular mass (B 2000 Da) displacers can be successfully employed for protein purification in ion-exchange displacement systems. These include dendrimers (Jayaraman et al., 1995), protected amino acids (Kundu et al., 1995a), antibiotics (Kundu et al., 1995b), and relatively nontoxic functionalized sucrose (Kundu et al., 1997). Despite the attractive advantages of displacement chromatography, there are no reports in the literature to date on the purification of recombinant protein therapeutics by displacement chromatography. In this manuscript we examine the utility of displacement chromatography for the purification of recombinant human brainderived neurotrophic factor, rHuBDNF. The rHuBDNF employed in this study was provided by Regeneron Pharmaceutical Inc. (Tarrytown, NY and Rensselaer, NY) as a partially purified mixture, pooled from an upstream chromatographic step. The protein is a dimer with an approximate molecular mass of 27 kDa. This growth factor is known to be quite basic, with a pI greater than 10. The rHuBDNF is expressed in E. coli and is harvested as an inclusion body. The purification of rHuBDNF by displacement chromatography is particularly challenging since rHuBDNF is very strongly bound to the cationexchange material. Furthermore, there are several variants of rHuBDNF which are extremely difficult to remove from the bioproduct of interest.
2. Materials and methods
2.1. Materials Regeneron Pharmaceutical Inc. provided the rHuBDNF feed stock used for the displacement
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experiments. Two different feed lots were employed. A feed stock of 0.4 mg ml − 1 protein concentration at 87% purity was utilized for the HS/M displacements at loadings per column volume of 2.1 and 11.0 mg ml − 1. All other experiments utilized a feed stock containing 1.25 mg ml − 1 protein at 87.5% purity (note: this is the same feed stock that was employed for the linear gradient process). Each feed stock was buffered with 50 mM sodium phosphate, pH 7.0, containing 275 mM NaCl. Protamine Sulfate Grade X from salmon (P4020, Lot 21H0519) was purchased from Sigma Chemical (St. Louis, MO, USA). Sodium Phosphate, phosphoric acid, acetonitrile, and TFA were purchased from Fisher Scientific (Pittsburgh, PA, USA). A POROS HS/M (4.6 ×100 mm) column used for initial displacer screening and bulk POROS HS/M resin were donated by PerSeptive Biosystems (Framingham, MA, USA). Displacement experiments were carried out using both 50 and 75 cm column lengths which were obtained by arranging 2 and 3 columns in series, respectively. A Vydac C4 (4.6× 250 mm) column was purchased from The Separations Group (Hisperia, CA, USA). OmegaChrom columns (0.46× 250 mm) were purchased from Upchurch Scientific (Oak Harbor, WA, USA).
2.2. Apparatus All displacement experiments were carried out using a Waters 625 LC system including a Waters 996 photodiode array detector, and a Waters Fraction Collector. Data collection and analysis was performed using Waters Millennium Chromatography Manager. Analysis of the collected fractions was carried out using a Waters 600S Gradient Controller, a Waters 626 Solvent Delivery Unit, a Waters PDA 996 photodiode array detector and a Waters 717 WISP autosampler with cooling module. A Waters Temperature Control Module was utilized for high-temperature reversed-phase analysis. Column packing was performed using PerSeptive Biosystems Self-Pack column packing apparatus.
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2.3. Analytical high-temperature RPLC Displacement fractions were analyzed using an acetonitrile gradient with a constant concentration of TFA as an ion-pairing agent. All analyses were performed using a Vydac C4 (4.6×250 mm) column at 65°C. Injection volume was adjusted to deliver 50 mg of total protein. The effluent was monitored at 215 nm.
2.4. Analytical ion-exchange chromatography The column feed stock was analyzed by ion-exchange chromatography using a 4.6× 250-mm columns packed with POROS HS50 resin. A gradient of sodium chloride was performed from 200 mM to 1200 mM over 10 column dead volumes. The mobile phase was buffered with 50 mM sodium phosphate, pH 7.0.
2.5. Displacement chromatography In all displacement experiments, the columns were initially equilibrated with the carrier and then sequentially perfused with feed, displacer and regenerant solutions. For all displacements, column loading was performed at a superficial mobile phase velocity of 180 cm h − 1 (0.50 ml min − 1) and the displacer was perfused at 90 cm h − 1 (0.25 ml min − 1). The mobile phase carrier was 50 mM sodium phosphate, 500 mM sodium chloride, pH 7.0. Displacer concentrations of 3 mM and 0.5 mM protamine sulfate were employed. Column lengths were 50 or 75 cm. The column effluent was monitored at 280 nm. Fraction sizes were 500 ml (for experiments using 3.0 mM protamine) and 1000 ml (for experiments using 0.5 mM protamine).
2.6. Column packing Two columns (4.6× 250 mm) of POROS HS/ M, three columns of (4.6×250 mm) of POROS HS50, and one (4.6× 50 mm) column of POROS HS50 were packed according to the following: Dry resin was slurried with packing buffer (50 mM sodium phosphate+ 150 mM sodium chloride) at a concentration of 0.1 g ml − 1. Packing
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was performed at constant velocity of 7200 cm h − 1 using the PerSeptive Self-Pack apparatus. Packing was complete after 150 column volumes of packing buffer were passed. After packing, each column was tested for asymmetry and plate count using a 20-ml pulse of 100 mM sodium nitrate as an unretained tracer with a mobile phase of 500 mM sodium chloride buffered at pH 7.0.
was first identified and used as a displacer by Gerstner (Gerstner and Cramer, 1992a). Protamine sulfate (5 kDa) is rich in arginine residues and possesses a high affinity for cation-exchange resins. Protamine sulfate itself has a therapeutic application as an antidote to the anticoagulant heparin (The United States Pharmacopeia, 1990).
3. Results and discussion
Initial displacement experiments were performed on an analytical (4.6× 500 mm) column packed with (20 mm) POROS HS/M resin. Fig. 2 presents a histogram of a displacement performed using 3 mM protamine displacer at a loading per column volume of 20.0 mg ml − 1, eight-times higher than used for the gradient process. As seen in the figure, protamine was able to produce an efficient displacement of the strongly bound rHuBDNF. Fractions from the column effluent were analyzed by RPLC and the resulting data were used to generate Fig. 3a-c. Although completely separated pure zones of product and impurities were not achieved, the impurities and product are found in highly enriched zones. This can be attributed to the difficulty of the separation. Difficult separations approach their final constant-pattern displacement profiles very slowly and may require long columns of impractical length (Antia and Horvath, 1991). Also, the finite degree of overlap that exists in constant-pattern displacement profiles (shock layer thickness) is also expected to be greater for very close separations (Zhu and Guiochon, 1994). Nevertheless, as seen in Fig. 3, the displacement process resulted in significant purification of the rHuBDNF with some fractions reaching as high as 96% purity. The product pool of all fractions greater than 85% purity (fractions 9 through 30) resulted in a 72.9% yield of product with a purity of 93.6% (note: this compares favorably to gradient chromatography which produced a yield of 67.9% at 93.1% purity at 8 times lower loadings). In addition to producing high yields and purity, the displacement process also resulted in an 8-fold concentration of the rHuBDNF to ap-
An analytical high temperature reversed-phase chromatogram of the rHuBDNF feed stream is shown in Fig. 1a. As seen in the figure, the feed consists of the rHuBDNF main peak along with several variants. The objective of this ion-exchange step was to reduce the variants below acceptable limits. Several ion-exchange resins were screened for their selectivity for this separation. The POROS HS resin (PerSeptive Biosystems, Framingham, MA) was found to have the highest selectivity for this separation and was observed to reproducibly reduce the variants to an acceptable level by linear gradient chromatography. However, in order to accomplish this separation, the linear gradient process was limited to low column loadings and very shallow linear gradients. Also seen in Fig. 1a are several later eluting impurity peaks that have been identified as host cell proteins. Removal of the host cell proteins is accomplished further downstream and is not the primary focus of the ion-exchange purification step. An analytical scale ion-exchange linear salt gradient was performed and the resulting chromatogram (Fig. 1b) reveals two difficulties encountered with this separation. First, the lack of baseline resolution or shoulders indicates that the protein variants have chromatographic properties very close to that of the bioproduct. Second, the rHuBDNF elutes at a very high mobile phase salt concentration (ca. 900 mM sodium chloride). This indicates that both the product and variants are strongly binding and implies the use of a very high affinity displacer. The displacer chosen for this work, protamine sulfate from salmon,
3.1. Displacements on POROS HS/M (20 mm) particles
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Fig. 1. Analytical chromatography of feed stock. (a) High-temperature reversed-phase acetonitrile gradient; Sample: 50 mg injection of rhuGFP ion-exchange feed; Column: 4.6 × 250 mm Vydac C4; (b) Analytical chromatogram on ion-exchange, pH 7.0; Sample: 20 mg of rhGFP ion-exchange feed; Column: 4.6 × 250 mm Poros HS50 strong cation-exchanger.
proximately 10 mg ml − 1. Thus, this displacement process resulted in simultaneous concentration and purification which is a well-established benefit of displacement mode chromatography (Horvath
and Brunner, 1985). Dynamic binding capacity data indicated that this displacement experiment was carried out at 33.6% of the total column capacity for this feed stock.
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Fig. 2. Histogram of ion-exchange displacement: 20 mg ml − 1 loading. Histogram showing total displaced protein (product and variants) and protamine sulfate displacer. Constructed from high-temperature reversed-phase analysis of displacement fractions.
Improved performance at high column loading is counterintuitive for practitioners of elution chromatography. In contrast to elution chromatography in which increased loading results in an increase in peak widths and hence overlap, increased loading in displacement chromatography merely increases the width of purified zones. Since in a developed displacement train the degree of overlap between zones is constant with respect to loading, increased loading can often improve pool purity, product yields and productivity. Results from two displacement experiments carried out at lower column loading illustrate this effect. A displacement experiment with a mass loading per column volume of 2.1 mg ml − 1 (similar to the loading of the linear gradient operation), resulted in a purity of 90.8% with a 62.5% yield. This purification lacked sufficient mass to achieve good recovery. By increasing the column loading to 11.0 mg ml − 1 the product purity and yield were increased to 92.8% and 72.6%, respectively. As described above, at an even higher loading of 20.0 mg ml − 1, the displacement con-
tinued to perform well, with concomitant increases in productivity. Analytical RPLC chromatograms of representative fractions found early, intermediate, and late in the 20 mg ml − 1 loading displacement experiment are presented in Fig. 4. While early displacement fractions are enriched in the variants found before the main peak, later fractions are enriched in variants found after the main product peak. These results indicate that the ion-exchange resin and the reversed-phase material appear to have a similar selectivity trend for the variants. This is unusual in that one would expect the selectivity of these two classes of resins to be quite different. Thus, these results indicate that the selectivity of the POROS ion-exchanger may be aided by nonspecific, hydrophobic interactions between the variants and the stationary phase material.
3.2. Displacements on POROS HS50 (50 mm) particles Purifications at bench-scale are often best performed using columns packed with small particle
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Fig. 3. Displacement fraction purity: 20 mg ml − 1 loading. Composition of fractions as determined by analytical high-temperature reversed-phase. Reported are rhuGFP main peak and variants as percent mass. (a) Feed and fractions 2 – 12; (b) Fractions 13 – 24; (c) Fractions 25 – 34.
resins due to their high efficiency and high capacity. In contrast, for manufacturing-scale chromatography, the performance benefits of smaller particle diameter resins are often outweighed by the drawbacks of higher pressure drop and resin cost. Since costly high pressure columns and
equipment would be required for performing process-scale chromatography with the POROS HS/ M resin, the utility of POROS HS50 (50 mm) resin for displacement chromatography was evaluated. A series of displacement experiments were carried out on the 50 mm material with a displacer
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Fig. 4. High temperature reversed-phase assay of three displacement fractions: 20 mg ml − 1 loading. Overlay of fractions collected from points early (fraction 16), intermediate (fraction 18), and late (fraction 31) of the displacement train. Note the variant enrichment in the early and late fractions.
concentration of 3.0 mM. These experiments did not result in acceptable reductions in the variants due to mass transport limitation inherent in the 50 mm material. Even when these experiments were carried out using longer column lengths (75 cm) the product pools were still below purity specifications. Overcoming the mass transport limitations of the 50 mm particles necessitated a six fold reduction in displacer concentration (to 0.5 mM) from that used with the 20 mm materials. A displacement experiment was carried out at a loading per column volume of 11.0 mg ml − 1 and at a displacer concentration of 0.5 mM. Fractions from the column effluent were analyzed by RPLC and
the fraction purity is presented in Fig. 5. As seen in the figure, the displacement process resulted in significant purification of the rHuBDNF with some fractions reaching as high as 95.4% purity. Pooling all fractions of greater than 85% purity (fractions 10 through 31) resulted in a 77.9% yield of product with a purity of 93.4%. Thus, by reducing the displacer concentration to 0.5 mM the results with the 50 mm materials became comparable to the best result with the 20 mm resin. Reducing the displacer concentration has several benefits. First, it is well established that reducing the displacer concentration lowers the concentrations of the product and impurity zones. These wider, less concentrated product zones can
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Fig. 5. Displacement fraction purity: 0.5 mM displacer concentration. Composition of fractions as determined by analytical high-temperature reversed-phase. Reported are rhGFP main peak and variant c 4 as percent mass. (a) Feed and fractions 3 – 13; (b) Fractions 14 – 25, (c) Fractions 26–35.
improve product yield due to a decrease in the relative amount of overlapping of enriched product and impurity zones, thereby diminishing the effects of mass transport limitations. Further, if desorption kinetics play a limiting role, slowing down the separation by reducing the displacer concentration may allow local equilibrium to be achieved.
3.3. Impacts on chromatographic producti6ity The production rate of rHuBDNF for the ionexchange step can be defined (Eq. (1)) in terms of the column loading of the rHuBDNF and its variants (mass per column volume), the yield of the step (Y), the purity of rHuBDNF in the feed
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Table 1 Cycle times computed for gradient and displacement purificationsa Mode/particle size
tequilibration (h)
tloading (h)
tseparation (h)
tregeneration (h)
tcycle (h)
Displacement/20 mm Displacement/50 mm Gradient/50 mm
4.0 4.0 4.0
16.0 8.8 1.9
4.5 13.2 11.0
22.1 22.1 23.6
46.7 48.2 40.5
a
Feed concentration and all volumetric flow rates (number of column volumes per hour) are equivalent for each purification.
(Pfeed), and the cycle time of the run (tcycle) (note: product purity and yield are defined with respect to the rHuBDNF and its variants; the small amount of host cell proteins were not included in these calculations) (Gallant et al., 1995, 1996). PR =
mfeed Vcolumn
YPfeed tcycle
(1)
Maximizing the production rate is an often sought after goal, as this allows the generation of the greatest amount of product in the least amount of time while using the least amount of stationary phase material. Increasing the production rate of a bottleneck purification step can impact the productivity of the entire purification process. The cycle time in Eq. (1) is the amount of time required for one complete cycle of the chromatographic step. This includes the time required for equilibration, loading, separation, and column regeneration. tcycle = tequilibration + tfeed +tseparation +tregeneration (2) The column regeneration time employed by the manufacturer for this process includes the time required for clean-in-place and sanitization procedures in addition to regeneration. Although it is more common to perform clean-in-place procedures after several cycles and to perform sanitization only between batches (Sofer and Nystrom, 1991), the current linear gradient process employs these aggressive measures to remove very strongly binding impurities and contaminants after each chromatographic use. Table 1 contains the various contributions to the cycle time obtained from the 20 mm displacement, the 50 mm displacement, and the linear gradient process for the same feed
lot. As seen in the table, while the equilibration and regeneration times remain relatively similar for these processes, the loading and separation times are quite different. The productivity of these separations is presented in Table 2. Both displacement processes resulted in significant improvements in the production rate. The 20 mm and 50 mm displacements resulted in an eight fold and 4.5 fold improvement, respectively, as compared to the current gradient process. It is worth noting that the production rate would be significantly higher if the inordinately long regeneration time were not employed. The 50 mm displacement provides a significant increase in production rate while utilizing existing manufacturing equipment. Without further optimization or equipment changes, the current practice of processing the ion-exchange feed in four separate sub-lots could be discontinued, thereby greatly impacting the productivity of the entire process. Other possible benefits include increased resin lifetime due to reduced exposure to harsh regenerating conditions and savings in labor costs. The increase in productivity of a 20-mm displacement can provide even greater savings of manufacturing time and resin volume but would require the added expense of high-pressure columns and equipment. A detailed economic analysis of the process is currently being performed to determine the practicality of using 20 mm resin in displacement mode. The cycle time of each displacement was strongly influenced by the loading time while the cycle time of the linear gradient was not (Table 1). Thus, an increase in the feed concentration brought about by improvements in upstream processes would result in further improvements to the
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Table 2 Productivity comparison of displacement and gradient elution purifications Mode/particle size
Loading (g l−1)
Feed purity (%) tcycle (h)
Yield (%)
Productivity (mg l−1 h−1)
Improvement w/r/t gradient
Displacement/20 mm Displacement/50 mm Gradient/50 mm
20.0
88.2
46.7
73.8
278.7
8.0x
11.0
87.1
48.2
77.9
155.0
4.5x
87.4
40.5
67.9
34.6
N/A
2.36
productivity of the displacement processes relative to the linear gradient process. The results presented in this paper demonstrate that displacement chromatography is capable of purifying rHuBDNF from its variants with both high yields and purity at elevated column loadings. Further, the displacement process provides a significant improvement in the productivity of this ion-exchange operation as compared to the linear gradient process.
Acknowledgements This research was supported by Grant No. GM47372 from the National Institutes of Health. The authors also wish to thank Dr Robert MacColl and Leslie Eisele of the Biochemistry Core at the Wadsworth Center, New York State Department of Health, for the use of their equipment and facilities.
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