- Email: [email protected]
High-performance tangential flow filtration using charged membranes
Journal of Membrane Science 159 (1999) 133±142
High-performance tangential ¯ow ®ltration using charged membranes R. van Reisa,*, J.M. Brakea, J. Charkoudianb, D.B. Burnsc, A.L. Zydneyc a
Separation Technology Group, Department of Recovery Sciences, Genentech, Inc., South San Francisco, CA 94080, USA b Department of Membrane Research, Millipore Corporation, Bedford, MA 01730, USA c Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA Received 14 September 1998; received in revised form 14 January 1999; accepted 20 January 1999
Abstract High-performance tangential ¯ow ®ltration (HPTFF) is an emerging technology that enables the separation of proteins with similar size. HPTFF technology has become possible by exploiting several new discoveries. It has been demonstrated that optimum selectivity and throughput are obtained in the pressure-dependent ¯ux regime. Selectivity and throughput can also be enhanced through module design and process con®gurations that reduce the transmembrane pressure gradient. Optimization of buffer pH and ionic strength have a signi®cant impact on the sieving behavior of proteins in membrane systems. Finally, a novel set of design equations and diagrams have been derived which provide a rational means for determining the optimum combination of selectivity and throughput for any given process. The current study focused on exploring the effects of membrane charge, in combination with buffer pH, on protein separation using HPTFF. Order-of-magnitude improvements in both selectivity and throughput were obtained by selecting the appropriate membrane charge at an optimum pH. The high selectivity and throughput values enabled protein puri®cation using a small number of diavolumes (N 4±12), reasonable membrane area per mass of product (17 m2 kgÿ1), and short processing times (1±3 h). Puri®cation factors up to 990-fold were obtained with yields of 94%. These results were obtained in linear scale-down systems representative of existing industrial scale systems, currently in use for ultra®ltration of human pharmaceuticals produced by recombinant DNA methods. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Biotechnology; Ultra®ltration; Protein; Puri®cation
1. Introduction HPTFF is an emerging technology that can be used to separate species with similar size using semipermeable membranes [1]. As originally described by van Reis [2,3], HPTFF obtained high selectivity by control of ®ltrate ¯ux and device ¯uid mechanics in order to *Corresponding author. Tel.: +1-650-225-1522; fax: +1-650225-4049; e-mail: [email protected]
minimize fouling and exploit the effects of concentration polarization. Increasing the concentration of a solute at the membrane wall increases the effective sieving of the solute in the absence of fouling. At higher wall concentrations fouling occurs, resulting in a reduction in the effective pore size. This, in turn, results in decreased sieving of the solute, despite the higher wall concentration. There is, therefore, an optimum ¯ux for separation of solutes using ultra®ltration membranes. This involves operating the mem-
0376-7388/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 9 9 ) 0 0 0 4 8 - 4
134
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
brane device in the pressure-dependent, rather than the pressure-independent, ¯ux regime. In addition, cocurrent ¯ow on the ®ltrate side of the membrane can be used to maintain the optimal ¯ux, and thus the maximum selectivity, throughout the module [2,3]. It was, subsequently, recognized that signi®cant improvements in performance could be obtained by controlling buffer pH and ionic strength to maximize differences in the effective hydrodynamic volume of the different proteins. For example, Saksena and Zydney [4] showed that the selectivity (de®ned as the ratio of the protein sieving coef®cients) for the ®ltration of bovine serum albumin (BSA) and immunoglobulin G (IgG) could be increased from a value of only two, at pH 7 and high salt concentrations, to more than 30 simply by adjusting the pH to 4.7 and lowering the solution ionic strength. The dramatic improvement in performance was due to the strong electrostatic exclusion of the positively charged IgG at pH 4.7, with the transmission of the (uncharged) BSA remaining fairly high. Similar improvements in performance by controlling pH and salt concentration have been reported for laboratory-scale ®ltration of BSA and hemoglobin [5], BSA and lysozyme [6], and myoglobin and cytochrome C [7], among others. van Reis et al. [1] demonstrated that this approach can be used for protein separation processes (BSA monomer±dimer and BSA±IgG) by using a dia®ltration mode to remove the more permeable species from the retained component. Even though these studies have demonstrated the importance of controlling electrostatic interactions in optimizing protein separations using HPTFF, there has been relatively little work on the possibility of exploiting the membrane charge to further enhance system performance. Miyama et al. [8] provided a preliminary analysis of protein ®ltration through both neutral and positively charged polyacrylonitrile membranes, with the latter produced by chemical modi®cation of the native polyacrylonitrile using a quaternary amine. BSA transmission through the positively charged membrane was signi®cantly greater than that through the base polyacrylonitrile at pH above the protein isoelectric point, i.e. under conditions where BSA was negatively charged. Miyama et al. [8] attributed this to attractive interactions between the negatively charged albumin and the positively charged membrane pores. Similar results were reported by Nakao
et al. [9] in a study using surface-modi®ed polysulfone membranes, with a negatively charged version produced by sulfonation and a positively charged version produced by chloromethylation, followed by quaternization of the amino group. Protein rejection was greatest under conditions where the membrane and protein had like charge due to strong electrostatic repulsion. Limited data obtained with mixtures of myoglobin and cytochrome C showed that the selectivity for this protein separation was also a function of the membrane charge. However, the best selectivity obtained in this study was only about ®ve, which is well below the selectivity required for an effective HPTFF process [10]. Millesime et al. [11,12] also studied the behavior of positively and negatively charged polysulfone membranes. Experimental data for BSA sieving at pH 7 (where BSA is negatively charged) showed a small increase in BSA transmission with increasing salt concentration for the negatively charged membrane, while BSA transmission through the positively charged membrane decreased with increasing salt concentration. In contrast, transmission of the positively charged lysozyme increased monotonically with increasing salt concentration for both, the positively and negatively charged membranes. Millesime et al. [11] hypothesized that this unusual behavior was due to the combination of protein fouling and direct electrostatic interactions, although no quantitative analysis of these phenomena was presented. Millesime et al. [12] subsequently examined the use of these positively charged membranes for the ®ltration of mixtures of BSA and lysozyme at pH 7. The positively charged membrane provided signi®cantly higher selectivity than the base polysulfone, even though the more permeable species (lysozyme) had the same charge as the membrane pores under these conditions. The higher selectivity was instead due to the strong rejection of the negatively charged BSA by the positively charged membrane, an effect which Millesime et al. [12] attributed to protein fouling. There was also evidence of intermolecular electrostatic interactions between the oppositely charged BSA and lysozyme at pH 7; however, these effects were not quanti®ed. Although these studies have demonstrated that membrane charge can have a signi®cant in¯uence on protein selectivity during HPTFF, there is still considerable uncertainty regarding both the magnitude and the origin of these effects.
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
The membrane charge appears to alter both the extent of membrane fouling and the magnitude of the electrostatic interactions between the protein and membrane pores. Furthermore, there is currently no clear understanding as to how to best exploit possible changes in the membrane charge as part of a more general strategy for the optimization of HPTFF processes. The objective of the current study was to quantitatively examine the effects of membrane charge, in combination with buffer pH, on protein separations using HPTFF. Filtration experiments were conducted with 100 kD polyethersulfone membranes with either negative or positive charge, as determined by streaming potential measurements. Data were obtained using a mixture of BSA and an antigen binding fragment (Fab) derived from a recombinant DNA antibody. These two proteins have similar molecular weight (68 kD for BSA and 45 kD for the Fab) but differ signi®cantly in their surface charge characteristics (the isoelectric point for BSA is 4.8 while that for the Fab is around 8.5). The effect of membrane charge and buffer pH on both selectivity and throughput were studied using total recycle experiments. Protein separation processes were then performed at selected optimal conditions using a dia®ltration mode to effectively remove the more permeable species. 2. Experimental 2.1. Proteins and buffer solutions The antigen binding fragment (Fab) of a recombinant DNA antibody was obtained from Genentech (South San Francisco, CA) as a 7.6-g lÿ1 solution in a pH 7, 0.5 M ammonium sulfate±10 mM sodium phosphate buffer. Sulfhydryl-modi®ed BSA was obtained from Bayer (Catalog Number 81-024, Kankakee, IL) and contained 70% monomer and 30% oligomer as determined by high-performance liquid chromatography (HPLC) size-exclusion chromatography (SEC). This BSA powder was dissolved in 10 mM, pH 7 phosphate buffer and puri®ed in an automated scale-down system using a two-stage closed loop HPTFF cascade [1] with a BiomaxTM 70-kD membrane (Millipore, Bedford, MA). The
135
resulting BSA was essentially 100% monomer. This solution was concentrated to 15.1 g/l by ultra®ltration with a 10-kD composite regenerated cellulose membrane in a PelliconTM XL cassette (Millipore, Bedford, MA) using a LabscaleTM TFF system (Millipore, Bedford, MA). HPTFF experiments were performed using acetate (pH 4.6±5.6) or tris (pH 8.0±8.7) buffers prepared from reagent grade sodium acetate (Sigma Chemical, St. Louis, MO), glacial acetic acid (Baker, Phillipsburg, NJ), and tris-base and tris-hydrochloride (Research Organics, Cleveland, OH). Solution ionic strength was set to 10 mM, with the pH then adjusted using the appropriate acid or base. Potassium hydrogen phthalate, potassium phosphate monobasic, and sodium borate (Fisher Scienti®c, Pittsburgh, PA) were used as buffers in the streaming potential experiments. The pH was measured using an Omega (Omega Engineering, Stamford, CT) or Acumet (Fisher Scienti®c, Pittsburgh, PA) pH probe. All buffers were pre®ltered through 0.22-mm cellulose acetate ®lters (either Corning, Corning, NY or Gelman Sciences, Ann Arbor, MI) prior to use. Mixtures of BSA monomer and Fab in acetate or tris buffer were prepared by dia®ltration using 7 diavolumes. Dia®ltration was performed with 10-kD composite regenerated cellulose membranes (PLCGC 10, Cat. # PXC010C50, Millipore, Bedford, MA) using a LabscaleTM TFF system (Millipore, Bedford, MA). The BSA±Fab solution was recovered from the reservoir, cassette, and retentate lines, and then ®ltered through a 0.22-mm SterivexTM GV ®lter (Cat. # SVGV01015, Millipore, Bedford, MA). 2.2. Protein assays All protein samples were assayed by HPLC±SEC using an automated system consisting of a pump, autosampler and injector, diode array detector, computer interface, and data-analysis software (Model 1100, Hewlett±Packard, Palo Alto, CA). SEC was performed using a SuperdexTM 200 column (10 mm inner diameter, 300 mm column length) obtained from Pharmacia Biotech (Piscataway, NJ). The sample injection volume was 75±100 ml and the mobile phase was 10 mM, pH 9 tris at a ¯ow rate of 0.75 ml/min. Peak detection was performed at 280 nm. Protein samples were also assayed by SDS±PAGE under non-reducing conditions using 10-well, 1-mm thick,
136
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
8±16% tris±glycine gels (Novex, San Diego, CA). The cathode and anode solutions were SDS running buffer. The gels were mounted in an Xcell gel electrophoresis cell (Novex, San Diego, CA) and run for 1 h at 125 V before staining with Coomasie brilliant blue. Low levels of BSA were also analyzed by ELISA, performed by the assay services department at Genentech (South San Francisco, CA), and having a detection limit of 0.1 ng/ml. 2.3. Protein charge Protein charge was estimated by calculating the net charge contributed from all the amino acids in the protein sequence as a function of pH. The effective net charge of the proteins will differ from this estimate since the tertiary structure of the protein will shield the charge of some amino acids, and the proteins may bind anions or cations from the buffer solution. 2.4. Membranes and membrane cleaning Protein separations were performed using BiomaxTM 100 negative membranes and prototype BiomaxTM 100 positive membranes, both provided by Millipore (Bedford, MA). Both the membranes have nominal molecular weight cut-off values of 100 000 Dalton. The BiomaxTM 100 positive membranes were produced by chemical modi®cation of the base polyethersulfone using a quaternary amine. The membranes were provided in PelliconTM XL cassettes having a nominal membrane area of 50 cm2 area. The PelliconTM XL modules are plate-and-frame style cassettes with a channel length of 15.7 cm and a channel width of 2.2 cm. The channel dimensions are larger than the active membrane area dimensions. The feed channel has Millipore type C screens between the membranes and a nominal channel height of 0.051 cm. The ®ltrate channels have Millipore type-B screens and a nominal channel height of 0.031 cm. BiomaxTM 100 negative membranes were cleaned with 0.052 weight % sodium hypochlorite (All Pure Chemical, Tracy, CA) at 508C for 30±60 min. BiomaxTM 100 positive membranes were cleaned in 0.02 N sodium hydroxide (Fisher, Fair Lawn, NJ) at pH 12 and ambient temperature for 60 min. Cleaning was done using total recycle at a feed ¯ow rate of
323 l m ÿ2 hÿ1 and a mean transmembrane pressure of 1.4 bar. All feed ¯ow rates are normalized with respect to membrane area. All membranes were cleaned before their ®rst use and then prior to each subsequent experiment to insure consistent performance. 2.5. Streaming potential measurements Membrane charge was evaluated from streaming potential measurements. The measurement device consisted of two Plexiglas chambers, each 2 cm in diameter and 2.4 cm in length. The ends of the chambers were threaded so that they could be screwed together to form a tight seal. Ag/AgCl electrodes were made by placing a 1-mm diameter silver wire (Sigma, St. Louis, MO) and a reducing electrode in a 1 M KCl solution. The wire and electrode were connected to a DC power source with the current maintained at 10 mA for 20 min. The Ag/AgCl electrodes were inserted through the ends of the chambers and sealed using O-rings to prevent leakage. Small circular membrane disks were cut from single large ¯at sheets. The device was assembled with the membrane carefully sealed between the two chambers. The chambers were ®lled with 10 mM KCl, buffered with 1 mM phthalate (pH 2.2±5.8), phosphate (pH 5.8±8), tris (pH 8±9), or borate (pH 9±10). The solution conductivity was measured using an ES-12 conductivity meter (Horiba, Kyoto, Japan) and adjusted to 1.47 mS/cm as needed. The device was pressurized by adjusting the height of the solution reservoir connected to one of the chambers. Flow from the opposite chamber was taken directly to drain. The system was allowed to equilibrate for at least 20 min, at which point the transmembrane voltage was measured using a 8060A True RMS Multimeter (Fluke, Everett, WA) with an accuracy of 0.01 mV. Data were obtained at several pressures, with the zeta potential evaluated from the slope of the voltage versus pressure data using the Helmholtz±Smoluchowski equation [13]. The zeta potential provides a measure of the electrostatic potential at the plane of shear, i.e. at the inner edge of the diffuse electrical double layer adjacent to the charged surface. After measuring the zeta potential at a given pH, the chambers and membrane were rinsed with deionized water and re-®lled with a fresh KCl solution to evaluate the zeta potential at a different pH.
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
Fig. 1. Schematic of filtration set-up used for HPTFF experiments. The filtrate was either returned to the feed tank for optimization experiments or was directed to a separate collection vessel for process separation experiments.
2.6. Filtration system HPTFF experiments were conducted using a LabscaleTM TFF system (Millipore, Bedford, MA) consisting of a 500-ml reservoir, stir base, retentate valve and retentate pressure guages, and connections for peristaltic pumps (Master¯ex1 L/STM Model 7519-10 Cartridge Pump heads, Master¯ex1 751955 tubing holders, and Master¯ex1 6±600 rpm console drive model 7521-40). A schematic of the HPTFF set-up used for optimization and process separation experiments is shown in Fig. 1. Filtrate ¯ow was controlled using a three-roller peristaltic Master¯ex1 QuickLoad1 pump head controlled by a Master¯ex1 pump drive (Model 7520-25). All pumps, pump heads, and tubing holders were obtained from Cole Parmer Instrument (Chicago, IL). Pressure was monitored by digital pressure gages (Model 68920-36, Psi Tronix, Tulare, CA). Feed and ®ltrate ¯ow rates were determined by timed collection using a PM4800 DeltaRange1 balance (Mettler, Hightstown, NJ). Initial HPTFF experiments were performed in the total recycle mode (returning both retentate and ®ltrate to the feed tank). The membrane was initially ¯ushed with 300 ml of buffer to remove any residual storage chemicals. The membrane hydraulic permeability was then evaluated from data
137
for the buffer ¯ux at several different transmembrane pressures. The feed reservoir was drained, keeping the lines and cassette ®lled. The protein solution was added to the feed reservoir, and the feed ¯ow rate was set to 323 l mÿ2 hÿ1 while maintaining zero transmembrane pressure. The retentate valve, ®ltrate pump, and co-current ¯ow pump were then manually adjusted to obtain equal transmembrane pressures at both the inlet and the outlet. Flux and pressure measurements were made after equilibration for a minimum of 10 min at each pressure. Samples of 500 ml of the feed and ®ltrate were obtained for subsequent protein assays. The retentate valve, co-current ¯ow pump, and ®ltrate pump were then re-adjusted to obtain a higher transmembrane pressure, taking care to insure operation in the pressure-dependent ¯ux regime. This was accomplished by tracking ¯ux versus transmembrane pressure curves during the experiments. HPTFF process separations were performed at selected optimal conditions of pH and ®ltrate ¯ux for each membrane as described in Section 3. The apparatus was the same as that used for the total recycle experiments, except that the ®ltrate was directed to a separate collection reservoir and a dia®ltration buffer tank was connected to the feed reservoir. Constant retentate volume was maintained by sealing the feed reservoir and allowing the vacuum generated by the ®ltrate pump to draw the dia®ltration buffer into the feed reservoir. The system was initially operated in total recycle mode for 15±20 min to obtain stable operation. The ®ltrate line was then removed from the feed reservoir and sent to a beaker for collection. Samples of 500 ml were obtained from the feed reservoir, the ®ltrate line, and ®ltrate collection reservoir every two diavolumes. 2.7. Data analysis Membrane separations can be optimized by using a set of equations that describe the process yield and puri®cation factor as a function of selectivity and throughput [10]. The dimensionless selectivity, , is de®ned as: S1 =S2
(1)
where S1 and S2 are the dimensionless observed sieving coef®cients for the less and more highly retained
138
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
solutes: S Cf =CF
(2)
where Cf is the ®ltrate concentration of a speci®c protein and CF the feed concentration of the same protein. A throughput parameter can be de®ned: JS J S1 ÿ S2 ÿ2
(3)
ÿ1
where J [l m h ] is the ®ltrate ¯ux. Alternatively, a dimensionless throughput parameter can be de®ned as: NS JS At=V
(4) 2
where N is the number of diavolumes, A (m ) the membrane area, t (h) the process time, and V (l) the retentate volume. The parameter JS can be directly measured as a function of ®ltrate ¯ux, solution pH, solution ionic strength, and membrane charge. The parameter NS includes the effects of membrane area and ®ltration time, and is thus the key parameter for actually designing an HPTFF system. However, the group At/V is often ca. 1 m2 h lÿ1 for a typical UF process, in which case JS NS with J having units of l mÿ2 hÿ1. The optimum combination of selectivity and throughput can be determined from the optimization diagrams which have previously been described [10]. Effective separations in HPTFF require large values of both and JS, with the selectivity determining the intrinsic separation capability while the throughput parameter de®nes the practical limits on the actual puri®cation that can be obtained in a commercial process.
3. Results and discussion 3.1. Membrane charge The charge of the BiomaxTM 100 negative and BiomaxTM 100 positive membranes as a function of pH was determined by streaming potential measurements. The results are shown in Fig. 2. The membrane isoelectric point, de®ned as the pH at which the measured zeta potential is zero, is around pH 2 for the BiomaxTM 100 negative membrane (determined by simple linear extrapolation of the zeta potential
Fig. 2. Membrane charge as a function of pH for the BiomaxTM 100 positive (*) and BiomaxTM 100 negative (*) membranes as determined by streaming potential measurements.
data). The BiomaxTM 100 positive membrane has an isoelectric point at a pH >9, as expected from the quaternary amine surface modi®cation. The BiomaxTM 100 positive membrane has an approximately constant charge throughout the pH 4.6±8.7 range used in the protein separation experiments, while the charge on the BiomaxTM 100 negative membrane becomes somewhat more negative with increasing pH. Further studies will be required to determine the effective charge after exposure to protein solutions at various pH values. 3.2. Selectivity and throughput Mixtures of 3 mg/ml BSA and 3 mg/ml Fab were prepared in 10 mM buffers in a pH 4.6±8.7 range. HPTFF sieving coef®cients for both the proteins were measured as a function of ¯ux in the range of 15± 90 l mÿ2 hÿ1. Selectivity and throughput values were calculated for each ¯ux set point. The resulting pairs of selectivity and throughput values were evaluated using optimization diagrams [10] to determine the best combination of these paired values. The selectivity values from this analysis are plotted as a function of pH in Fig. 3. The y-axis shows the selectivity for BSA ( BSA SFab/SBSA when BSA is the more highly retained solute), and the selectivity for Fab ( Fab SBSA/SFab when the Fab is the more highly retained solute), both on positive logarithmic scales with an intercept ( 1), where there is no preferential selectivity for either protein (SBSA SFab). The
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
139
Fig. 3. Selectivity for BSA ( BSA) and Fab ( Fab) in BSA±Fab mixtures as a function of pH for the BiomaxTM 100 positive (*) and BiomaxTM 100 negative (*) membranes.
Fig. 4. Throughput for BSA (JSBSA) and Fab (JSFab) in BSA± Fab mixtures as a function of pH for the BiomaxTM 100 positive (*) and BiomaxTM 100 negative (*) membranes.
highest selectivity for BSA was obtained at pH 8.4 (close to the Fab pI 8.5) using a negatively charged membrane at a ¯ux of 55 l mÿ2 hÿ1. The BSA was almost completely rejected under these conditions (S 0.004) while the Fab was able to pass relatively freely through the membrane (S 0.8), corresponding to a selectivity of 200. The positively charged membrane had more than an order of magnitude lower selectivity under these same pH conditions, which was primarily due to an increase in BSA transmission (S 0.15), while the Fab sieving coef®cient was unchanged. Conversely, the highest selectivity for Fab was obtained at pH 5.0 (close to the BSA pI 4.8) using a positively charged membrane at a ¯ux of 62 l mÿ2 hÿ1. In this case, the Fab sieving coef®cient was S 0.006 while the BSA sieving coef®cient was S 0.7, nearly a complete reversal of the behavior seen at pH 8.4 with the negatively charged membrane. Again, an order of magnitude reduction in selectivity was obtained when using the negatively charged membrane with the pH 5.0 buffer. These data clearly indicate that the protein sieving coef®cient is dramatically reduced when the protein is highly charged (i.e. at a pH away from the pI) and has the same sign of the charge as the membrane, conditions which maximize the protein's effective volume [14] while enhancing the electrostatic exclusion associated with direct charge±charge interactions [15]. Sieving is enhanced when the pH is near the protein pI, i.e. where the effective hydrodynamic volume is minimal. The selectivity was only a
weak function of pH near the pI of the less retained protein. This may be due to the fact that the protein will have patches of positive and negative charges, both above and below the pI despite the fact that the net charge changes sign. The throughput values corresponding to the selectivity data are shown in Fig. 4. The maximum values of JS were obtained around pH 8.4 for the negatively charged membrane and around pH 5.0 for the positively charged membrane, paralleling the behavior seen in Fig. 3 for the selectivity. Order-of-magnitude improvements in both
Fig. 5. Order-of-magnitude improvements in both selectivity ( ) and throughput (JS) are obtained by selecting the appropriate membrane charge at an optimum buffer pH. Selectivity and throughput are plotted for BSA/Fab and Fab/BSA separations using BiomaxTM 100 negative (*) and BiomaxTM 100 positive (*) membranes at pH 5.0 and pH 8.4.
140
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
HPTFF process experiments for BSA/Fab and Fab/ BSA separations were designed based on the optimum membrane charge, pH, and ¯ux conditions derived from the initial sieving experiments. Solutions of 3 mg/ml BSA and 3 mg/ml Fab were prepared in either a 10-mM tris buffer at pH 8.4 or a 10-mM acetate buffer at pH 5.0. The pH 8.4 protein solution was dia®ltered with a BiomaxTM 100 negative membrane at a feed rate of 323 l mÿ2 hÿ1 and a constant ®ltrate ¯ux of 44 l mÿ2 hÿ1. The pH 5.0 protein solution was dia®ltered with a BiomaxTM 100 positive membrane at a feed rate of 323 l mÿ2 hÿ1 and a constant ®ltrate ¯ux of 63 l mÿ2 hÿ1. A Coomasie stained non-reducing SDS±PAGE gel of retentate samples taken throughout the pH 8.4 dia®ltration process is shown in Fig. 6. The gel shows that the majority of the Fab is removed within four dia± volumes of dia®ltration. The gel electrophoresis analysis was complemented by HPLC analysis to determine the exact level of puri®cation. Analysis
of yield and puri®cation factor at the end of the process using the optimization equations presented by van Reis and Saksena [10] indicated average selectivity and throughput values of 110 and JS 23 l mÿ2 hÿ1 compared to 200 and JS 44 l mÿ2 hÿ1 for the total recycle experiments. Replicate experiments con®rmed the higher selectivity (standard deviation of 10%) at the initial bulk concentrations. Further studies are required to elucidate the cause of these changes in selectivity and throughput. Despite the reduced selectivity and throughput, the pH 8.4 HPTFF process with the BiomaxTM 100 negative membrane resulted in 94% BSA yield with 990-fold puri®cation in 3.1 h using 13 diavolumes of buffer. A Coomasie stained non-reducing SDS-PAGE gel of retentate samples taken throughout the pH 5.0 dia®ltration process is shown in Fig. 7. The gel shows that the majority of the BSA is removed within six diavolumes of dia®ltration. The gel electrophoresis analysis was complemented by HPLC and ELISA analysis to determine the exact level of puri®cation. Analysis of yield and puri®cation factor at the end of the process indicated average selectivity and throughput values of 19 and JS 35 l mÿ2 hÿ1 compared to 110 and JS 44 l mÿ2 hÿ1 for the total recycle experiments. Replicate experiments con®rmed the higher selectivity (standard deviation of 11%) at the initial bulk concentrations. The pH 5.0 HPTFF process with the BiomaxTM 100 positive membrane resulted in 69% Fab yield with 830-fold puri®cation in 2.8 h using 12 diavolumes of buffer.
Fig. 6. Electrophoretic gel of BSA±Fab mixture before, and during, HPTFF purification process performed with BiomaxTM 100 negative membrane at pH 8.4. N, number of diavolumes. The main upper band is BSA and the main lower band is Fab.
Fig. 7. Electrophoretic gel of BSA±Fab mixture before, and during, HPTFF purification process performed with BiomaxTM 100 positive membrane at pH 5.0. N, number of diavolumes. The main upper band is BSA and the main lower band is Fab.
selectivity and throughput, can hence be obtained by selecting the appropriate membrane charge at an optimal pH for a given separation. These results are summarized in Fig. 5. The combination of high selectivity and high JS values provides the opportunity to conduct effective protein separations using a combination of membrane area, number of diavolumes, and processing time that is typical of conventional ultra®ltration processes. 3.3. Protein purification
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
4. Conclusions This study combines important discoveries in membrane science to enable HPTFF processes with high yield and puri®cation factors. Operating in the pressure-dependent ¯ux regime [2,3], selectively altering protein charge through buffer pH [4], exploiting membrane charge effects [9], and using optimization diagrams [10], has resulted in orderof-magnitude improvements in HPTFF performance. This study provides the clearest demonstration to-date of the signi®cant effects of membrane charge on HPTFF separations, and also provides important insight into the nature of electrostatic interactions controlling transmission of charged proteins through charged membranes. Further optimization should be possible by optimizing the feed ¯ow rate, ionic strength, bulk protein concentration, and the membrane pore size. All of these parameters were kept constant in this study. The membrane pore size and pore-size distribution should have a particularly pronounced effect. Optimization of feed ¯ow rate [1] and ionic strength [4] has previously been described. Based on this study and previous work in the ®eld, it is now possible to formulate some simple guidelines for HPTFF process development. Two-dimensional isoelectric focusing provides a convenient analysis of the molecular weight and charge distribution of product protein and impurities. Based on these data, one can select one or several sequential pH values for dia®ltration. The pH values should be close to the pI values of the impurities (pH pI 1) and should be as far away from the pI of the product protein as possible to maximize the difference in effective hydrodynamic volume of the components. The ionic strength should be as low as possible but suf®cient to provide buffering capacity and to ensure protein stability [A reasonable starting point is 10 mM.] Operating at low ionic strength near the pI of the impurities may pose some limitations. These conditions can cause protein denaturation and precipitation and subsequent membrane fouling. Stabilization of proteins using non-ionic solutes should, henceforth, be considered to maintain high selectivity and throughput values. The membrane charge should be of the same sign as the charge on the product protein at the selected pH value to enhance the electrostatic exclu-
141
sion of the product from the membrane pores. Membrane pore size must be chosen based on experience. In this study, it was possible to obtain excellent performance with a single choice of pore size. If initial experiments give a low selectivity value one should try a tighter pore size. If the throughput value is too low one should test a larger pore size. It should be possible to optimize an HPTFF process with no more than two membrane pore-size trials. A single feed ¯ow rate can be chosen for initial experiments and may be optimized if required at a later stage. Experiments are performed to determine selectivity and JS as a function of pH, ¯ux, and bulk protein concentration. The best combination of selectivity and JS are then determined using the optimization diagrams [10]. Yield and puri®cation goals can now be combined with membrane area and process time requirements. Finally, process runs are performed to test actual process performance. Industrial applications of HPTFF will often involve complex mixtures with several impurities of varying molecular weight and isoelectric point. It may, hence, be desirable to perform multiple dia®ltration steps with different buffer pH values or to generate a pH gradient during dia®ltration. The very high JS values demonstrated in this study make it feasible to do this with reasonable membrane areas and process times. On account of the very high selectivity and JS values obtained in this study, it was possible to achieve up to 990-fold puri®cation with 94% yields using process parameters that are typical for industrial scale protein concentration and buffer exchange [16]. All HPTFF experiments in this study were carried out with linear scale-down equipment [17]. The feed ¯ow rate (323 l mÿ2 hÿ1), membrane area-to-mass ratio (16± 18 m2/kg), number of diavolumes (4±13), and process time (3.1 h) are all representative of industrial-scale processes. HPTFF technology can provide protein concentration, buffer exchange and puri®cation in a single unit operation. These processes are currently performed using a combination of 2±3 separate chromatography and ultra®ltration steps. HPTFF technology, hence, has the potential to be utilized as a universal high throughput process with reduced production costs. In light of demonstrated high throughput values, HPTFF can be considered for both initial and ®nal puri®cation stages analogous to the use of conventional ultra®ltration.
142
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
5. List of symbols A BSA Cf CF Fab HPLC HPTFF IgG J kD N pI S SEC t TFF UF V
membrane area (m2) bovine serum albumin filtrate concentration (g/l) feed concentration (g/l) antigen binding fragment of antibody high-performance liquid chromatography high-performance tangential flow filtration immunoglobulin G filtrate flux (l mÿ2 hÿ1) kilo-Dalton number of diavolumes isoelectric point sieving coefficient size-exclusion chromatography process time (h) tangential flow filtration ultrafiltration retentate volume (l) selectivity
Acknowledgements The authors gratefully acknowledge Tracy Perkins, Philip Lester, Debbie Ansaldi, Jimmy Sugahara, and Kevin Ng (Genentech, Inc.) for puri®cation of BSA monomer and Fab, Adeyma Arroyo (Genentech, Inc.) for assay support, and Phil Goddard, Mark Chisholm, Ralf Kuriyel, and Steven Pearl (Millipore Corporation) for providing prototype modules and project support. References [1] R. van Reis, S. Gadam, L.N. Frautschy, S. Orlando, E.M. Goodrich, S. Saksena, R. Kuriyel, C.M. Simpson, S. Pearl, A.L. Zydney, High performance tangential flow filtration, Biotech. Bioeng. 56 (1997) 71±82.
[2] R. van Reis, US. Patent 5,256,294. Tangential Flow Filtration Process and Apparatus (1993). [3] R. van Reis, US Patent 5,490,937. Tangential Flow Filtration Process and Apparatus (1996). [4] S. Saksena, A.L. Zydney, Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective membrane filtration, Biotech. Bioeng. 43 (1994) 960±968. [5] H.C.M. van Eijndhoven, S. Saksena, A.L. Zydney, Protein fractionation using membrane filtration: role of electrostatic interactions, Biotech. Bioeng. 48 (1995) 406±414. [6] E. Iritani, Y. Mukai, T. Murase, Upward dead-end ultrafiltration of binary protein mixtures, Sep. Sci. Tech. 30 (1995) 369±382. [7] M.-C. Yang, J.-H. Tong, Loose ultrafiltration of proteins using hydrolyzed polyacrylonitrile hollow fiber, J. Membrane Sci. 132 (1997) 63±71. [8] H. Miyama, K. Tanaka, Y. Nosaka, N. Fujii, H. Tanzawa, S. Nagaoka, Charged ultrafiltration membrane for permeation of proteins, J. Appl. Polymer Sci. 36 (1988) 925±933. [9] S. Nakao, H. Osada, H. Kurata, T. Tsuru, S. Kimura, Separation of proteins by charged ultrafiltration membranes, Desalination 70 (1988) 191±205. [10] R. van Reis, S. Saksena, Optimization diagram for membrane separations, J. Membrane Sci. 129 (1997) 19±29. [11] L. Millesime, C. Amiel, B. Chaufer, Ultrafiltration of lysozyme and bovine serum albumin with polysulfone membranes modified with quaternized polyvinylimidazole, J. Membrane Sci. 89 (1994) 223±234. [12] L. Millesime, J. Dulieu, B. Chaufer, Fractionation of proteins with modified membranes, Bioseparation 6 (1996) 135± 145. [13] M. NystroÈm, M. LindstroÈm, E. Matthiasson, Streaming potential as a tool in the characterization of ultrafiltration membranes, Coll. Surf. 36 (1989) 297±306. [14] N.S. Pujar, A.L. Zydney, Electrostatic effects on protein partitioning in size exclusion chromatography and membrane ultrafiltration, J. Chromatogr. A 796 (1998) 229±238. [15] N.S. Pujar, A.L. Zydney, Charge regulation and electrostatic interactions for a spherical particle in a cylindrical pore, J. Coll. Interface Sci. 192 (1997) 338±349. [16] R. van Reis, E.M. Goodrich, C.L. Yson, L.N. Frautschy, R. Whiteley, A.L. Zydney, Constant C wall ultrafiltration process control, J. Membrane Sci. 130 (1997) 123±140. [17] R. van Reis, E.M. Goodrich, C.L. Yson, L.N. Frautschy, S. Dzengeleski, H. Lutz, Linear scale ultrafiltration, Biotech. Bioeng. 55 (1997) 737±746.
High-performance tangential ¯ow ®ltration using charged membranes R. van Reisa,*, J.M. Brakea, J. Charkoudianb, D.B. Burnsc, A.L. Zydneyc a
Separation Technology Group, Department of Recovery Sciences, Genentech, Inc., South San Francisco, CA 94080, USA b Department of Membrane Research, Millipore Corporation, Bedford, MA 01730, USA c Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA Received 14 September 1998; received in revised form 14 January 1999; accepted 20 January 1999
Abstract High-performance tangential ¯ow ®ltration (HPTFF) is an emerging technology that enables the separation of proteins with similar size. HPTFF technology has become possible by exploiting several new discoveries. It has been demonstrated that optimum selectivity and throughput are obtained in the pressure-dependent ¯ux regime. Selectivity and throughput can also be enhanced through module design and process con®gurations that reduce the transmembrane pressure gradient. Optimization of buffer pH and ionic strength have a signi®cant impact on the sieving behavior of proteins in membrane systems. Finally, a novel set of design equations and diagrams have been derived which provide a rational means for determining the optimum combination of selectivity and throughput for any given process. The current study focused on exploring the effects of membrane charge, in combination with buffer pH, on protein separation using HPTFF. Order-of-magnitude improvements in both selectivity and throughput were obtained by selecting the appropriate membrane charge at an optimum pH. The high selectivity and throughput values enabled protein puri®cation using a small number of diavolumes (N 4±12), reasonable membrane area per mass of product (17 m2 kgÿ1), and short processing times (1±3 h). Puri®cation factors up to 990-fold were obtained with yields of 94%. These results were obtained in linear scale-down systems representative of existing industrial scale systems, currently in use for ultra®ltration of human pharmaceuticals produced by recombinant DNA methods. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Biotechnology; Ultra®ltration; Protein; Puri®cation
1. Introduction HPTFF is an emerging technology that can be used to separate species with similar size using semipermeable membranes [1]. As originally described by van Reis [2,3], HPTFF obtained high selectivity by control of ®ltrate ¯ux and device ¯uid mechanics in order to *Corresponding author. Tel.: +1-650-225-1522; fax: +1-650225-4049; e-mail: [email protected]
minimize fouling and exploit the effects of concentration polarization. Increasing the concentration of a solute at the membrane wall increases the effective sieving of the solute in the absence of fouling. At higher wall concentrations fouling occurs, resulting in a reduction in the effective pore size. This, in turn, results in decreased sieving of the solute, despite the higher wall concentration. There is, therefore, an optimum ¯ux for separation of solutes using ultra®ltration membranes. This involves operating the mem-
0376-7388/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 9 9 ) 0 0 0 4 8 - 4
134
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
brane device in the pressure-dependent, rather than the pressure-independent, ¯ux regime. In addition, cocurrent ¯ow on the ®ltrate side of the membrane can be used to maintain the optimal ¯ux, and thus the maximum selectivity, throughout the module [2,3]. It was, subsequently, recognized that signi®cant improvements in performance could be obtained by controlling buffer pH and ionic strength to maximize differences in the effective hydrodynamic volume of the different proteins. For example, Saksena and Zydney [4] showed that the selectivity (de®ned as the ratio of the protein sieving coef®cients) for the ®ltration of bovine serum albumin (BSA) and immunoglobulin G (IgG) could be increased from a value of only two, at pH 7 and high salt concentrations, to more than 30 simply by adjusting the pH to 4.7 and lowering the solution ionic strength. The dramatic improvement in performance was due to the strong electrostatic exclusion of the positively charged IgG at pH 4.7, with the transmission of the (uncharged) BSA remaining fairly high. Similar improvements in performance by controlling pH and salt concentration have been reported for laboratory-scale ®ltration of BSA and hemoglobin [5], BSA and lysozyme [6], and myoglobin and cytochrome C [7], among others. van Reis et al. [1] demonstrated that this approach can be used for protein separation processes (BSA monomer±dimer and BSA±IgG) by using a dia®ltration mode to remove the more permeable species from the retained component. Even though these studies have demonstrated the importance of controlling electrostatic interactions in optimizing protein separations using HPTFF, there has been relatively little work on the possibility of exploiting the membrane charge to further enhance system performance. Miyama et al. [8] provided a preliminary analysis of protein ®ltration through both neutral and positively charged polyacrylonitrile membranes, with the latter produced by chemical modi®cation of the native polyacrylonitrile using a quaternary amine. BSA transmission through the positively charged membrane was signi®cantly greater than that through the base polyacrylonitrile at pH above the protein isoelectric point, i.e. under conditions where BSA was negatively charged. Miyama et al. [8] attributed this to attractive interactions between the negatively charged albumin and the positively charged membrane pores. Similar results were reported by Nakao
et al. [9] in a study using surface-modi®ed polysulfone membranes, with a negatively charged version produced by sulfonation and a positively charged version produced by chloromethylation, followed by quaternization of the amino group. Protein rejection was greatest under conditions where the membrane and protein had like charge due to strong electrostatic repulsion. Limited data obtained with mixtures of myoglobin and cytochrome C showed that the selectivity for this protein separation was also a function of the membrane charge. However, the best selectivity obtained in this study was only about ®ve, which is well below the selectivity required for an effective HPTFF process [10]. Millesime et al. [11,12] also studied the behavior of positively and negatively charged polysulfone membranes. Experimental data for BSA sieving at pH 7 (where BSA is negatively charged) showed a small increase in BSA transmission with increasing salt concentration for the negatively charged membrane, while BSA transmission through the positively charged membrane decreased with increasing salt concentration. In contrast, transmission of the positively charged lysozyme increased monotonically with increasing salt concentration for both, the positively and negatively charged membranes. Millesime et al. [11] hypothesized that this unusual behavior was due to the combination of protein fouling and direct electrostatic interactions, although no quantitative analysis of these phenomena was presented. Millesime et al. [12] subsequently examined the use of these positively charged membranes for the ®ltration of mixtures of BSA and lysozyme at pH 7. The positively charged membrane provided signi®cantly higher selectivity than the base polysulfone, even though the more permeable species (lysozyme) had the same charge as the membrane pores under these conditions. The higher selectivity was instead due to the strong rejection of the negatively charged BSA by the positively charged membrane, an effect which Millesime et al. [12] attributed to protein fouling. There was also evidence of intermolecular electrostatic interactions between the oppositely charged BSA and lysozyme at pH 7; however, these effects were not quanti®ed. Although these studies have demonstrated that membrane charge can have a signi®cant in¯uence on protein selectivity during HPTFF, there is still considerable uncertainty regarding both the magnitude and the origin of these effects.
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
The membrane charge appears to alter both the extent of membrane fouling and the magnitude of the electrostatic interactions between the protein and membrane pores. Furthermore, there is currently no clear understanding as to how to best exploit possible changes in the membrane charge as part of a more general strategy for the optimization of HPTFF processes. The objective of the current study was to quantitatively examine the effects of membrane charge, in combination with buffer pH, on protein separations using HPTFF. Filtration experiments were conducted with 100 kD polyethersulfone membranes with either negative or positive charge, as determined by streaming potential measurements. Data were obtained using a mixture of BSA and an antigen binding fragment (Fab) derived from a recombinant DNA antibody. These two proteins have similar molecular weight (68 kD for BSA and 45 kD for the Fab) but differ signi®cantly in their surface charge characteristics (the isoelectric point for BSA is 4.8 while that for the Fab is around 8.5). The effect of membrane charge and buffer pH on both selectivity and throughput were studied using total recycle experiments. Protein separation processes were then performed at selected optimal conditions using a dia®ltration mode to effectively remove the more permeable species. 2. Experimental 2.1. Proteins and buffer solutions The antigen binding fragment (Fab) of a recombinant DNA antibody was obtained from Genentech (South San Francisco, CA) as a 7.6-g lÿ1 solution in a pH 7, 0.5 M ammonium sulfate±10 mM sodium phosphate buffer. Sulfhydryl-modi®ed BSA was obtained from Bayer (Catalog Number 81-024, Kankakee, IL) and contained 70% monomer and 30% oligomer as determined by high-performance liquid chromatography (HPLC) size-exclusion chromatography (SEC). This BSA powder was dissolved in 10 mM, pH 7 phosphate buffer and puri®ed in an automated scale-down system using a two-stage closed loop HPTFF cascade [1] with a BiomaxTM 70-kD membrane (Millipore, Bedford, MA). The
135
resulting BSA was essentially 100% monomer. This solution was concentrated to 15.1 g/l by ultra®ltration with a 10-kD composite regenerated cellulose membrane in a PelliconTM XL cassette (Millipore, Bedford, MA) using a LabscaleTM TFF system (Millipore, Bedford, MA). HPTFF experiments were performed using acetate (pH 4.6±5.6) or tris (pH 8.0±8.7) buffers prepared from reagent grade sodium acetate (Sigma Chemical, St. Louis, MO), glacial acetic acid (Baker, Phillipsburg, NJ), and tris-base and tris-hydrochloride (Research Organics, Cleveland, OH). Solution ionic strength was set to 10 mM, with the pH then adjusted using the appropriate acid or base. Potassium hydrogen phthalate, potassium phosphate monobasic, and sodium borate (Fisher Scienti®c, Pittsburgh, PA) were used as buffers in the streaming potential experiments. The pH was measured using an Omega (Omega Engineering, Stamford, CT) or Acumet (Fisher Scienti®c, Pittsburgh, PA) pH probe. All buffers were pre®ltered through 0.22-mm cellulose acetate ®lters (either Corning, Corning, NY or Gelman Sciences, Ann Arbor, MI) prior to use. Mixtures of BSA monomer and Fab in acetate or tris buffer were prepared by dia®ltration using 7 diavolumes. Dia®ltration was performed with 10-kD composite regenerated cellulose membranes (PLCGC 10, Cat. # PXC010C50, Millipore, Bedford, MA) using a LabscaleTM TFF system (Millipore, Bedford, MA). The BSA±Fab solution was recovered from the reservoir, cassette, and retentate lines, and then ®ltered through a 0.22-mm SterivexTM GV ®lter (Cat. # SVGV01015, Millipore, Bedford, MA). 2.2. Protein assays All protein samples were assayed by HPLC±SEC using an automated system consisting of a pump, autosampler and injector, diode array detector, computer interface, and data-analysis software (Model 1100, Hewlett±Packard, Palo Alto, CA). SEC was performed using a SuperdexTM 200 column (10 mm inner diameter, 300 mm column length) obtained from Pharmacia Biotech (Piscataway, NJ). The sample injection volume was 75±100 ml and the mobile phase was 10 mM, pH 9 tris at a ¯ow rate of 0.75 ml/min. Peak detection was performed at 280 nm. Protein samples were also assayed by SDS±PAGE under non-reducing conditions using 10-well, 1-mm thick,
136
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
8±16% tris±glycine gels (Novex, San Diego, CA). The cathode and anode solutions were SDS running buffer. The gels were mounted in an Xcell gel electrophoresis cell (Novex, San Diego, CA) and run for 1 h at 125 V before staining with Coomasie brilliant blue. Low levels of BSA were also analyzed by ELISA, performed by the assay services department at Genentech (South San Francisco, CA), and having a detection limit of 0.1 ng/ml. 2.3. Protein charge Protein charge was estimated by calculating the net charge contributed from all the amino acids in the protein sequence as a function of pH. The effective net charge of the proteins will differ from this estimate since the tertiary structure of the protein will shield the charge of some amino acids, and the proteins may bind anions or cations from the buffer solution. 2.4. Membranes and membrane cleaning Protein separations were performed using BiomaxTM 100 negative membranes and prototype BiomaxTM 100 positive membranes, both provided by Millipore (Bedford, MA). Both the membranes have nominal molecular weight cut-off values of 100 000 Dalton. The BiomaxTM 100 positive membranes were produced by chemical modi®cation of the base polyethersulfone using a quaternary amine. The membranes were provided in PelliconTM XL cassettes having a nominal membrane area of 50 cm2 area. The PelliconTM XL modules are plate-and-frame style cassettes with a channel length of 15.7 cm and a channel width of 2.2 cm. The channel dimensions are larger than the active membrane area dimensions. The feed channel has Millipore type C screens between the membranes and a nominal channel height of 0.051 cm. The ®ltrate channels have Millipore type-B screens and a nominal channel height of 0.031 cm. BiomaxTM 100 negative membranes were cleaned with 0.052 weight % sodium hypochlorite (All Pure Chemical, Tracy, CA) at 508C for 30±60 min. BiomaxTM 100 positive membranes were cleaned in 0.02 N sodium hydroxide (Fisher, Fair Lawn, NJ) at pH 12 and ambient temperature for 60 min. Cleaning was done using total recycle at a feed ¯ow rate of
323 l m ÿ2 hÿ1 and a mean transmembrane pressure of 1.4 bar. All feed ¯ow rates are normalized with respect to membrane area. All membranes were cleaned before their ®rst use and then prior to each subsequent experiment to insure consistent performance. 2.5. Streaming potential measurements Membrane charge was evaluated from streaming potential measurements. The measurement device consisted of two Plexiglas chambers, each 2 cm in diameter and 2.4 cm in length. The ends of the chambers were threaded so that they could be screwed together to form a tight seal. Ag/AgCl electrodes were made by placing a 1-mm diameter silver wire (Sigma, St. Louis, MO) and a reducing electrode in a 1 M KCl solution. The wire and electrode were connected to a DC power source with the current maintained at 10 mA for 20 min. The Ag/AgCl electrodes were inserted through the ends of the chambers and sealed using O-rings to prevent leakage. Small circular membrane disks were cut from single large ¯at sheets. The device was assembled with the membrane carefully sealed between the two chambers. The chambers were ®lled with 10 mM KCl, buffered with 1 mM phthalate (pH 2.2±5.8), phosphate (pH 5.8±8), tris (pH 8±9), or borate (pH 9±10). The solution conductivity was measured using an ES-12 conductivity meter (Horiba, Kyoto, Japan) and adjusted to 1.47 mS/cm as needed. The device was pressurized by adjusting the height of the solution reservoir connected to one of the chambers. Flow from the opposite chamber was taken directly to drain. The system was allowed to equilibrate for at least 20 min, at which point the transmembrane voltage was measured using a 8060A True RMS Multimeter (Fluke, Everett, WA) with an accuracy of 0.01 mV. Data were obtained at several pressures, with the zeta potential evaluated from the slope of the voltage versus pressure data using the Helmholtz±Smoluchowski equation [13]. The zeta potential provides a measure of the electrostatic potential at the plane of shear, i.e. at the inner edge of the diffuse electrical double layer adjacent to the charged surface. After measuring the zeta potential at a given pH, the chambers and membrane were rinsed with deionized water and re-®lled with a fresh KCl solution to evaluate the zeta potential at a different pH.
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
Fig. 1. Schematic of filtration set-up used for HPTFF experiments. The filtrate was either returned to the feed tank for optimization experiments or was directed to a separate collection vessel for process separation experiments.
2.6. Filtration system HPTFF experiments were conducted using a LabscaleTM TFF system (Millipore, Bedford, MA) consisting of a 500-ml reservoir, stir base, retentate valve and retentate pressure guages, and connections for peristaltic pumps (Master¯ex1 L/STM Model 7519-10 Cartridge Pump heads, Master¯ex1 751955 tubing holders, and Master¯ex1 6±600 rpm console drive model 7521-40). A schematic of the HPTFF set-up used for optimization and process separation experiments is shown in Fig. 1. Filtrate ¯ow was controlled using a three-roller peristaltic Master¯ex1 QuickLoad1 pump head controlled by a Master¯ex1 pump drive (Model 7520-25). All pumps, pump heads, and tubing holders were obtained from Cole Parmer Instrument (Chicago, IL). Pressure was monitored by digital pressure gages (Model 68920-36, Psi Tronix, Tulare, CA). Feed and ®ltrate ¯ow rates were determined by timed collection using a PM4800 DeltaRange1 balance (Mettler, Hightstown, NJ). Initial HPTFF experiments were performed in the total recycle mode (returning both retentate and ®ltrate to the feed tank). The membrane was initially ¯ushed with 300 ml of buffer to remove any residual storage chemicals. The membrane hydraulic permeability was then evaluated from data
137
for the buffer ¯ux at several different transmembrane pressures. The feed reservoir was drained, keeping the lines and cassette ®lled. The protein solution was added to the feed reservoir, and the feed ¯ow rate was set to 323 l mÿ2 hÿ1 while maintaining zero transmembrane pressure. The retentate valve, ®ltrate pump, and co-current ¯ow pump were then manually adjusted to obtain equal transmembrane pressures at both the inlet and the outlet. Flux and pressure measurements were made after equilibration for a minimum of 10 min at each pressure. Samples of 500 ml of the feed and ®ltrate were obtained for subsequent protein assays. The retentate valve, co-current ¯ow pump, and ®ltrate pump were then re-adjusted to obtain a higher transmembrane pressure, taking care to insure operation in the pressure-dependent ¯ux regime. This was accomplished by tracking ¯ux versus transmembrane pressure curves during the experiments. HPTFF process separations were performed at selected optimal conditions of pH and ®ltrate ¯ux for each membrane as described in Section 3. The apparatus was the same as that used for the total recycle experiments, except that the ®ltrate was directed to a separate collection reservoir and a dia®ltration buffer tank was connected to the feed reservoir. Constant retentate volume was maintained by sealing the feed reservoir and allowing the vacuum generated by the ®ltrate pump to draw the dia®ltration buffer into the feed reservoir. The system was initially operated in total recycle mode for 15±20 min to obtain stable operation. The ®ltrate line was then removed from the feed reservoir and sent to a beaker for collection. Samples of 500 ml were obtained from the feed reservoir, the ®ltrate line, and ®ltrate collection reservoir every two diavolumes. 2.7. Data analysis Membrane separations can be optimized by using a set of equations that describe the process yield and puri®cation factor as a function of selectivity and throughput [10]. The dimensionless selectivity, , is de®ned as: S1 =S2
(1)
where S1 and S2 are the dimensionless observed sieving coef®cients for the less and more highly retained
138
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
solutes: S Cf =CF
(2)
where Cf is the ®ltrate concentration of a speci®c protein and CF the feed concentration of the same protein. A throughput parameter can be de®ned: JS J S1 ÿ S2 ÿ2
(3)
ÿ1
where J [l m h ] is the ®ltrate ¯ux. Alternatively, a dimensionless throughput parameter can be de®ned as: NS JS At=V
(4) 2
where N is the number of diavolumes, A (m ) the membrane area, t (h) the process time, and V (l) the retentate volume. The parameter JS can be directly measured as a function of ®ltrate ¯ux, solution pH, solution ionic strength, and membrane charge. The parameter NS includes the effects of membrane area and ®ltration time, and is thus the key parameter for actually designing an HPTFF system. However, the group At/V is often ca. 1 m2 h lÿ1 for a typical UF process, in which case JS NS with J having units of l mÿ2 hÿ1. The optimum combination of selectivity and throughput can be determined from the optimization diagrams which have previously been described [10]. Effective separations in HPTFF require large values of both and JS, with the selectivity determining the intrinsic separation capability while the throughput parameter de®nes the practical limits on the actual puri®cation that can be obtained in a commercial process.
3. Results and discussion 3.1. Membrane charge The charge of the BiomaxTM 100 negative and BiomaxTM 100 positive membranes as a function of pH was determined by streaming potential measurements. The results are shown in Fig. 2. The membrane isoelectric point, de®ned as the pH at which the measured zeta potential is zero, is around pH 2 for the BiomaxTM 100 negative membrane (determined by simple linear extrapolation of the zeta potential
Fig. 2. Membrane charge as a function of pH for the BiomaxTM 100 positive (*) and BiomaxTM 100 negative (*) membranes as determined by streaming potential measurements.
data). The BiomaxTM 100 positive membrane has an isoelectric point at a pH >9, as expected from the quaternary amine surface modi®cation. The BiomaxTM 100 positive membrane has an approximately constant charge throughout the pH 4.6±8.7 range used in the protein separation experiments, while the charge on the BiomaxTM 100 negative membrane becomes somewhat more negative with increasing pH. Further studies will be required to determine the effective charge after exposure to protein solutions at various pH values. 3.2. Selectivity and throughput Mixtures of 3 mg/ml BSA and 3 mg/ml Fab were prepared in 10 mM buffers in a pH 4.6±8.7 range. HPTFF sieving coef®cients for both the proteins were measured as a function of ¯ux in the range of 15± 90 l mÿ2 hÿ1. Selectivity and throughput values were calculated for each ¯ux set point. The resulting pairs of selectivity and throughput values were evaluated using optimization diagrams [10] to determine the best combination of these paired values. The selectivity values from this analysis are plotted as a function of pH in Fig. 3. The y-axis shows the selectivity for BSA ( BSA SFab/SBSA when BSA is the more highly retained solute), and the selectivity for Fab ( Fab SBSA/SFab when the Fab is the more highly retained solute), both on positive logarithmic scales with an intercept ( 1), where there is no preferential selectivity for either protein (SBSA SFab). The
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
139
Fig. 3. Selectivity for BSA ( BSA) and Fab ( Fab) in BSA±Fab mixtures as a function of pH for the BiomaxTM 100 positive (*) and BiomaxTM 100 negative (*) membranes.
Fig. 4. Throughput for BSA (JSBSA) and Fab (JSFab) in BSA± Fab mixtures as a function of pH for the BiomaxTM 100 positive (*) and BiomaxTM 100 negative (*) membranes.
highest selectivity for BSA was obtained at pH 8.4 (close to the Fab pI 8.5) using a negatively charged membrane at a ¯ux of 55 l mÿ2 hÿ1. The BSA was almost completely rejected under these conditions (S 0.004) while the Fab was able to pass relatively freely through the membrane (S 0.8), corresponding to a selectivity of 200. The positively charged membrane had more than an order of magnitude lower selectivity under these same pH conditions, which was primarily due to an increase in BSA transmission (S 0.15), while the Fab sieving coef®cient was unchanged. Conversely, the highest selectivity for Fab was obtained at pH 5.0 (close to the BSA pI 4.8) using a positively charged membrane at a ¯ux of 62 l mÿ2 hÿ1. In this case, the Fab sieving coef®cient was S 0.006 while the BSA sieving coef®cient was S 0.7, nearly a complete reversal of the behavior seen at pH 8.4 with the negatively charged membrane. Again, an order of magnitude reduction in selectivity was obtained when using the negatively charged membrane with the pH 5.0 buffer. These data clearly indicate that the protein sieving coef®cient is dramatically reduced when the protein is highly charged (i.e. at a pH away from the pI) and has the same sign of the charge as the membrane, conditions which maximize the protein's effective volume [14] while enhancing the electrostatic exclusion associated with direct charge±charge interactions [15]. Sieving is enhanced when the pH is near the protein pI, i.e. where the effective hydrodynamic volume is minimal. The selectivity was only a
weak function of pH near the pI of the less retained protein. This may be due to the fact that the protein will have patches of positive and negative charges, both above and below the pI despite the fact that the net charge changes sign. The throughput values corresponding to the selectivity data are shown in Fig. 4. The maximum values of JS were obtained around pH 8.4 for the negatively charged membrane and around pH 5.0 for the positively charged membrane, paralleling the behavior seen in Fig. 3 for the selectivity. Order-of-magnitude improvements in both
Fig. 5. Order-of-magnitude improvements in both selectivity ( ) and throughput (JS) are obtained by selecting the appropriate membrane charge at an optimum buffer pH. Selectivity and throughput are plotted for BSA/Fab and Fab/BSA separations using BiomaxTM 100 negative (*) and BiomaxTM 100 positive (*) membranes at pH 5.0 and pH 8.4.
140
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
HPTFF process experiments for BSA/Fab and Fab/ BSA separations were designed based on the optimum membrane charge, pH, and ¯ux conditions derived from the initial sieving experiments. Solutions of 3 mg/ml BSA and 3 mg/ml Fab were prepared in either a 10-mM tris buffer at pH 8.4 or a 10-mM acetate buffer at pH 5.0. The pH 8.4 protein solution was dia®ltered with a BiomaxTM 100 negative membrane at a feed rate of 323 l mÿ2 hÿ1 and a constant ®ltrate ¯ux of 44 l mÿ2 hÿ1. The pH 5.0 protein solution was dia®ltered with a BiomaxTM 100 positive membrane at a feed rate of 323 l mÿ2 hÿ1 and a constant ®ltrate ¯ux of 63 l mÿ2 hÿ1. A Coomasie stained non-reducing SDS±PAGE gel of retentate samples taken throughout the pH 8.4 dia®ltration process is shown in Fig. 6. The gel shows that the majority of the Fab is removed within four dia± volumes of dia®ltration. The gel electrophoresis analysis was complemented by HPLC analysis to determine the exact level of puri®cation. Analysis
of yield and puri®cation factor at the end of the process using the optimization equations presented by van Reis and Saksena [10] indicated average selectivity and throughput values of 110 and JS 23 l mÿ2 hÿ1 compared to 200 and JS 44 l mÿ2 hÿ1 for the total recycle experiments. Replicate experiments con®rmed the higher selectivity (standard deviation of 10%) at the initial bulk concentrations. Further studies are required to elucidate the cause of these changes in selectivity and throughput. Despite the reduced selectivity and throughput, the pH 8.4 HPTFF process with the BiomaxTM 100 negative membrane resulted in 94% BSA yield with 990-fold puri®cation in 3.1 h using 13 diavolumes of buffer. A Coomasie stained non-reducing SDS-PAGE gel of retentate samples taken throughout the pH 5.0 dia®ltration process is shown in Fig. 7. The gel shows that the majority of the BSA is removed within six diavolumes of dia®ltration. The gel electrophoresis analysis was complemented by HPLC and ELISA analysis to determine the exact level of puri®cation. Analysis of yield and puri®cation factor at the end of the process indicated average selectivity and throughput values of 19 and JS 35 l mÿ2 hÿ1 compared to 110 and JS 44 l mÿ2 hÿ1 for the total recycle experiments. Replicate experiments con®rmed the higher selectivity (standard deviation of 11%) at the initial bulk concentrations. The pH 5.0 HPTFF process with the BiomaxTM 100 positive membrane resulted in 69% Fab yield with 830-fold puri®cation in 2.8 h using 12 diavolumes of buffer.
Fig. 6. Electrophoretic gel of BSA±Fab mixture before, and during, HPTFF purification process performed with BiomaxTM 100 negative membrane at pH 8.4. N, number of diavolumes. The main upper band is BSA and the main lower band is Fab.
Fig. 7. Electrophoretic gel of BSA±Fab mixture before, and during, HPTFF purification process performed with BiomaxTM 100 positive membrane at pH 5.0. N, number of diavolumes. The main upper band is BSA and the main lower band is Fab.
selectivity and throughput, can hence be obtained by selecting the appropriate membrane charge at an optimal pH for a given separation. These results are summarized in Fig. 5. The combination of high selectivity and high JS values provides the opportunity to conduct effective protein separations using a combination of membrane area, number of diavolumes, and processing time that is typical of conventional ultra®ltration processes. 3.3. Protein purification
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
4. Conclusions This study combines important discoveries in membrane science to enable HPTFF processes with high yield and puri®cation factors. Operating in the pressure-dependent ¯ux regime [2,3], selectively altering protein charge through buffer pH [4], exploiting membrane charge effects [9], and using optimization diagrams [10], has resulted in orderof-magnitude improvements in HPTFF performance. This study provides the clearest demonstration to-date of the signi®cant effects of membrane charge on HPTFF separations, and also provides important insight into the nature of electrostatic interactions controlling transmission of charged proteins through charged membranes. Further optimization should be possible by optimizing the feed ¯ow rate, ionic strength, bulk protein concentration, and the membrane pore size. All of these parameters were kept constant in this study. The membrane pore size and pore-size distribution should have a particularly pronounced effect. Optimization of feed ¯ow rate [1] and ionic strength [4] has previously been described. Based on this study and previous work in the ®eld, it is now possible to formulate some simple guidelines for HPTFF process development. Two-dimensional isoelectric focusing provides a convenient analysis of the molecular weight and charge distribution of product protein and impurities. Based on these data, one can select one or several sequential pH values for dia®ltration. The pH values should be close to the pI values of the impurities (pH pI 1) and should be as far away from the pI of the product protein as possible to maximize the difference in effective hydrodynamic volume of the components. The ionic strength should be as low as possible but suf®cient to provide buffering capacity and to ensure protein stability [A reasonable starting point is 10 mM.] Operating at low ionic strength near the pI of the impurities may pose some limitations. These conditions can cause protein denaturation and precipitation and subsequent membrane fouling. Stabilization of proteins using non-ionic solutes should, henceforth, be considered to maintain high selectivity and throughput values. The membrane charge should be of the same sign as the charge on the product protein at the selected pH value to enhance the electrostatic exclu-
141
sion of the product from the membrane pores. Membrane pore size must be chosen based on experience. In this study, it was possible to obtain excellent performance with a single choice of pore size. If initial experiments give a low selectivity value one should try a tighter pore size. If the throughput value is too low one should test a larger pore size. It should be possible to optimize an HPTFF process with no more than two membrane pore-size trials. A single feed ¯ow rate can be chosen for initial experiments and may be optimized if required at a later stage. Experiments are performed to determine selectivity and JS as a function of pH, ¯ux, and bulk protein concentration. The best combination of selectivity and JS are then determined using the optimization diagrams [10]. Yield and puri®cation goals can now be combined with membrane area and process time requirements. Finally, process runs are performed to test actual process performance. Industrial applications of HPTFF will often involve complex mixtures with several impurities of varying molecular weight and isoelectric point. It may, hence, be desirable to perform multiple dia®ltration steps with different buffer pH values or to generate a pH gradient during dia®ltration. The very high JS values demonstrated in this study make it feasible to do this with reasonable membrane areas and process times. On account of the very high selectivity and JS values obtained in this study, it was possible to achieve up to 990-fold puri®cation with 94% yields using process parameters that are typical for industrial scale protein concentration and buffer exchange [16]. All HPTFF experiments in this study were carried out with linear scale-down equipment [17]. The feed ¯ow rate (323 l mÿ2 hÿ1), membrane area-to-mass ratio (16± 18 m2/kg), number of diavolumes (4±13), and process time (3.1 h) are all representative of industrial-scale processes. HPTFF technology can provide protein concentration, buffer exchange and puri®cation in a single unit operation. These processes are currently performed using a combination of 2±3 separate chromatography and ultra®ltration steps. HPTFF technology, hence, has the potential to be utilized as a universal high throughput process with reduced production costs. In light of demonstrated high throughput values, HPTFF can be considered for both initial and ®nal puri®cation stages analogous to the use of conventional ultra®ltration.
142
R. van Reis et al. / Journal of Membrane Science 159 (1999) 133±142
5. List of symbols A BSA Cf CF Fab HPLC HPTFF IgG J kD N pI S SEC t TFF UF V
membrane area (m2) bovine serum albumin filtrate concentration (g/l) feed concentration (g/l) antigen binding fragment of antibody high-performance liquid chromatography high-performance tangential flow filtration immunoglobulin G filtrate flux (l mÿ2 hÿ1) kilo-Dalton number of diavolumes isoelectric point sieving coefficient size-exclusion chromatography process time (h) tangential flow filtration ultrafiltration retentate volume (l) selectivity
Acknowledgements The authors gratefully acknowledge Tracy Perkins, Philip Lester, Debbie Ansaldi, Jimmy Sugahara, and Kevin Ng (Genentech, Inc.) for puri®cation of BSA monomer and Fab, Adeyma Arroyo (Genentech, Inc.) for assay support, and Phil Goddard, Mark Chisholm, Ralf Kuriyel, and Steven Pearl (Millipore Corporation) for providing prototype modules and project support. References [1] R. van Reis, S. Gadam, L.N. Frautschy, S. Orlando, E.M. Goodrich, S. Saksena, R. Kuriyel, C.M. Simpson, S. Pearl, A.L. Zydney, High performance tangential flow filtration, Biotech. Bioeng. 56 (1997) 71±82.
[2] R. van Reis, US. Patent 5,256,294. Tangential Flow Filtration Process and Apparatus (1993). [3] R. van Reis, US Patent 5,490,937. Tangential Flow Filtration Process and Apparatus (1996). [4] S. Saksena, A.L. Zydney, Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective membrane filtration, Biotech. Bioeng. 43 (1994) 960±968. [5] H.C.M. van Eijndhoven, S. Saksena, A.L. Zydney, Protein fractionation using membrane filtration: role of electrostatic interactions, Biotech. Bioeng. 48 (1995) 406±414. [6] E. Iritani, Y. Mukai, T. Murase, Upward dead-end ultrafiltration of binary protein mixtures, Sep. Sci. Tech. 30 (1995) 369±382. [7] M.-C. Yang, J.-H. Tong, Loose ultrafiltration of proteins using hydrolyzed polyacrylonitrile hollow fiber, J. Membrane Sci. 132 (1997) 63±71. [8] H. Miyama, K. Tanaka, Y. Nosaka, N. Fujii, H. Tanzawa, S. Nagaoka, Charged ultrafiltration membrane for permeation of proteins, J. Appl. Polymer Sci. 36 (1988) 925±933. [9] S. Nakao, H. Osada, H. Kurata, T. Tsuru, S. Kimura, Separation of proteins by charged ultrafiltration membranes, Desalination 70 (1988) 191±205. [10] R. van Reis, S. Saksena, Optimization diagram for membrane separations, J. Membrane Sci. 129 (1997) 19±29. [11] L. Millesime, C. Amiel, B. Chaufer, Ultrafiltration of lysozyme and bovine serum albumin with polysulfone membranes modified with quaternized polyvinylimidazole, J. Membrane Sci. 89 (1994) 223±234. [12] L. Millesime, J. Dulieu, B. Chaufer, Fractionation of proteins with modified membranes, Bioseparation 6 (1996) 135± 145. [13] M. NystroÈm, M. LindstroÈm, E. Matthiasson, Streaming potential as a tool in the characterization of ultrafiltration membranes, Coll. Surf. 36 (1989) 297±306. [14] N.S. Pujar, A.L. Zydney, Electrostatic effects on protein partitioning in size exclusion chromatography and membrane ultrafiltration, J. Chromatogr. A 796 (1998) 229±238. [15] N.S. Pujar, A.L. Zydney, Charge regulation and electrostatic interactions for a spherical particle in a cylindrical pore, J. Coll. Interface Sci. 192 (1997) 338±349. [16] R. van Reis, E.M. Goodrich, C.L. Yson, L.N. Frautschy, R. Whiteley, A.L. Zydney, Constant C wall ultrafiltration process control, J. Membrane Sci. 130 (1997) 123±140. [17] R. van Reis, E.M. Goodrich, C.L. Yson, L.N. Frautschy, S. Dzengeleski, H. Lutz, Linear scale ultrafiltration, Biotech. Bioeng. 55 (1997) 737±746.