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Ind. Eng. Chem. Res. 2006, 45, 1058-1063
SEPARATIONS Comparison of Alternative Chelex Carousel Processes for Zinc Removal from a Protein Solution Sungyong Mun* Department of Chemical Engineering, Hanyang UniVersity, Seoul, 133-791, Korea
Nien-Hwa Linda Wang School of Chemical Engineering, Purdue UniVersity, West Lafayette, Indiana 47907
Chelex 100 serves as a highly effective stationary phase for the separation of metal ions from proteins. A three-zone Chelex carousel process with four columns was developed previously for the removal of Zn ions from protein in a buffer solution. In this study, alternative Chelex carousel processes with different zone configurations and zone numbers were developed to improve one or more of the economic factors in the previous three-zone Chelex carousel process. To increase the throughput per bed volume, the regeneration and the re-equilibration zone can be combined into one zone. To reduce the product dilution and increase the throughput per bed volume, the length of the loading zone can be increased or the washing step can be moved out of the loading zone into a separate zone. Comparison of the alternative carousel processes showed that a two-zone carousel with three columns in the loading zone is the most economical for zinc removal from protein. 1. Introduction Chelex 100 has a high affinity for metal ions, whereas proteins with a molecular weight of 1000 or higher are totally excluded from the particle pores. For this reason, Chelex 100 has received much attention as a highly effective ion-exchange resin for the separation of metal ions from proteins.1-13 In previous studies,14 a three-zone carousel process based on Chelex 100 has been developed for the removal of zinc ions from a protein in 1 N acetic acid. Figure 1 shows the schematic diagram of the previous threezone Chelex carousel process, where each port moves periodically in the same direction as fluid flow to simulate periodic column movement countercurrent to the fluid flow. The function of each zone in Figure 1 is as follows: (1) zone I is used for re-equilibration of a column with the same eluent as in the separation step, to prepare the column for the separation task; (2) zone II is used for the regeneration of a column, i.e., the removal of all the zinc ions on the resin with a strong acid; (3) zone III is used for the separation of zinc ions from protein (product). The three-zone carousel is operated in the following manner (see Figure 1). In step N, the feed, which contains zinc ions and protein, is loaded into the first column (C) of zone III. During the feed loading, most of the zinc ions are adsorbed onto the resin, whereas protein molecules are excluded. After the feed loading, the columns (C and D) in zone III are washed such that column C should be free of protein before column switching (see Figure 1). Note that the loading and washing steps occur in series within every switching period, and the zinc ion adsorption wave should be confined within zone III * To whom correspondence should be addressed. Tel.: +82-2-22200483. Fax: +82-2-2298-4101. E-mail:
[email protected].
Figure 1. Schematic of the three-zone Chelex carousel process with four columns for the removal of zinc from protein.
throughout the loading and washing periods. When the end of protein trailing wave exits column C, step N + 1 begins; the feed inlet is switched to column D, which becomes the first
10.1021/ie050955w CCC: $33.50 © 2006 American Chemical Society Published on Web 01/04/2006
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column in zone III. Column A, which has been in the re-equilibration zone in step N, moves into zone III and becomes the second column of zone III. Meanwhile, column C, which has been loaded with zinc ions during step N, moves into the regeneration zone (zone II). Column B, which has been regenerated during step N, moves into the re-equilibration zone (zone I). The system is operated continuously in this cyclic manner. For the aforementioned carousel process, operating parameters such as zone flow rates and switching time were optimized for the highest throughput in a previous study.14 On the other hand, system parameters such as the number of columns in each zone (i.e., zone configuration) and the number of zones were fixed in all the designs and experiments.14 However, economic factors such as throughput, mobile phase consumption, product dilution, and production rate are affected by both the operating parameters and the system parameters associated with each zone, which should be optimized according to a given objective. The goal of this study is to develop alternative Chelex carousel processes with different zone configurations and zone numbers, which can lead to further improvements in one or more of the aforementioned economic factors. The developed carousel processes are then compared in terms of the economic factors.
For the mobile phase, the mass-transfer equation for each solute i includes contributions from convection, axial dispersion, and film mass transfer:
∂Cm,i ∂2Cm,i ∂Cm,i 3kf,i(1 - b) ) Eb,i 2 - u0 (Cm,i - Cp,i|r)Rp) ∂t ∂z Rpb ∂z (7a) z ) zk,0, Eb,i
z ) zk,L,
Zn2+ + 2R-H+ S 2H+ + R2-Zn2+
(1)
CH3COOH S CH3COO- + H+
(2)
where R- represents the fixed negatively charged ionic sites on Chelex 100. The positively charged ions, Zn2+ and H+, are the counterions. Acetic acid undergoes a partial dissociation reaction, as shown in eq 2. The above two reactions result in the following equilibrium relations and constraints.
C h ZnCH2 CZnC h H2
) KZn,H
CHCCH3COO CCH3COOH
(ion-exchange equilibrium)
(3)
(dissociation equilibrium)
(4)
) Ka
(7c)
p
( )
∂qi ∂Cp,i Dp,i ∂ 2∂Cp,i r + (1 - p) ) p 2 ∂t ∂t ∂r r ∂r r ) 0,
∂Cp,i )0 ∂r
∂Cp,i ) kf,i(Cm,i - Cp,i|r)Rp) r ) Rp, pDp,i ∂r
qi )
C hi (1 - b)(1 - p)
(electroneutrality in ion exchanger) (6)
where the subscript “m” represents the mobile phase.
where Ci (expressed in units of equiv/L) is the concentration of component i in the solution phase and C h i (given in units of equiv/L of packed bed) is the concentration of component i on the resin phase. KZn,H is the mass action equilibrium constant. Ka is the dissociation equilibrium constant of acetic acid.
(8b) (8c)
(9)
Because the mass-transfer processes and the ion-exchange and dissociation reactions are coupled, the aforementioned relations and equations (eqs 1-8) are solved simultaneously in a versatile reaction and separation (VERSE) simulator. The VERSE simulator has been validated in several previous studies.15-24 2.2. Nonreacting Species (Protein). Protein molecules are totally excluded from the resin particles; thus, there is no need to consider the mass-transfer equation for the pore phase. Only the mass-transfer equation for the mobile phase is needed for modeling the migration behavior of protein in a carousel ionexchanger. The equation includes contributions from only convection and axial dispersion, as follows:
(5)
(electroneutrality in solution)
(8a)
where Cp,i is the pore-phase concentration, qi is the solid-phase concentration (presented in units of equiv/L of solid volume), r is the distance in the radial direction, and Dp,i is the pore (intraparticle) diffusivity. Note that qi in eq 8a is related to C hi in eq 3 as follows:
∂2Cm,protein ∂Cm,protein ∂Cm,protein - u0 ) Eb,insulin 2 ∂t ∂z ∂z
CZn + CH ) CCH3COO + CCl
C h Zn + C hH ) C hT
∂Cm,i )0 ∂z
(7b)
where Cm,i is the mobile-phase concentration of species i at time t and axial position z, Cf,i represents the inlet concentration of species i, Eb,i is the axial dispersion coefficient of species i, zk,0 represents the axial position of the inlet of column k, zk,L represents the axial position of the outlet of column k, u0 is the mobile-phase interstitial velocity, kf,i is the film mass-transfer coefficient, Rp is the particle radius, and Cp,i|r)Rp is the solidphase concentration at the surface of a solid particle. In the pore phase, the mass-transfer equation includes contributions from adsorption and Fickian diffusion:
2. Mathematical Model The ion-exchange and dissociation reactions occur in the Chelex carousel system.14 Besides the two reactions, a series of transport processes of each species in the mobile and pore phases form the foundation of the mathematical model for analyzing a carousel ion-exchange process. The key equations for the reactions and the transport processes are briefly discussed below. 2.1. Reacting Species. In the system of interest, the ionexchange and dissociation reactions occur between zinc chloride (ZnCl2) dissolved in 1 N acetic acid and Chelex 100 in the hydrogen form.14 Five species (such as Zn2+, Cl-, CH3COOH, CH3COO-, and H+) participate in the two reactions, as follows:
∂Cm,i ) u0(Cm,i(t,zk,0) - Cf,i(t,zk,0)) ∂z
(10)
3. Approach The three-zone Chelex carousel with four columns (see Figure 1), which was developed in a previous study,14 can be modified to improve its efficiency. To increase the throughput per bed
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Ind. Eng. Chem. Res., Vol. 45, No. 3, 2006 Table 1. Operating Conditions of the Previous Three-Zone Chelex Carousel Process with Four Columnsa parameter
value
zone configuration zone flow rates (mL/min) zone I zone II zone III inlet flow rates (mL/min) feed wash switching time (min) loading time (min) washing time (min)
1-1-2 10.0 9.0 4.0 4.0 4.0 28 21 7
a Column dimension: 1.1 cm ID × 30 cm length. The extra-column dead volume is 1.13 mL per column, or 4.0% of the bed volume.
Figure 2. Two-zone Chelex carousel process with three columns (Strategy A): (a) process diagram, and (b) steady-state column profiles at the end of a switching period from VERSE simulations.
volume, one can combine the regeneration and the re-equilibration zone into one zone (Strategy A). To reduce the product dilution and increase the throughput per bed volume, one can increase the length of the loading zone (Strategy B) or move the washing step out of the loading zone into a separate zone (Strategy C). The proposed carousel processes, which are based on the aforementioned strategies, are validated with VERSE simulations and then compared in terms of the economic factors while the product purity and yield are fixed at 100% and 99.5%, respectively. The intrinsic parameters used in the VERSE simulations were reported in a previous study.14 4. Results and Discussion 4.1. Two-Zone Chelex Carousel with Three Columns (Strategy A). In Strategy A, a two-zone carousel with three columns (zone configuration: 1-2; see Figure 2a) is proposed. The loading zone (zone II) of this process has the same number of columns as that of the previous three-zone carousel;14 therefore, there is no difference in the switching time, washing time, and loading time between the two processes, as shown in Tables 1 and 2. The only difference is that the proposed process uses only one zone (zone I) for both regeneration and reequilibration. This, in turn, increases the zone I flow rate, because both regeneration and re-equilibration should be completed within one switching time (see Table 2). The steadystate column profiles for this process are obtained from VERSE simulations and are shown in Figure 2b. This strategy has a higher throughput per bed volume than the previous three-zone carousel process (Table 3). 4.2. Two-Zone Chelex Carousel with Four Columns (Strategy B). In Strategy B, a two-zone carousel with four columns (zone configuration: 1-3; see Figure 3a) is proposed. This process has three columns in the loading zone (zone II), unlike the aforementioned two-zone carousel with two columns in the loading zone. The longer loading zone allows a longer loading time, while the washing time was kept the same (see
Table 2). As the loading time increases, the amount of protein injected into the feed port increases accordingly. The increase in the amount of protein compensates more than the amount of mobile phase consumed during the extended loading time. For this reason, the proposed process with three columns in the loading zone leads to less mobile-phase consumption (expressed in terms of the number of liters of mobile phase per kilogram of protein) and less product dilution than the other two carousels with two columns in the loading zone (see Table 3). The column profiles of the proposed process with three columns in the loading zone are obtained from VERSE simulations at the end of a switching period when the system reaches a steady state. As shown in Figure 3b, the zinc adsorption wave is confined within the loading zone while the protein trailing wave completely exits the first column in the loading zone. These results prove that high purity and a high yield of protein are maintained throughout the carousel operation. 4.3. Chelex Carousel without a Washing Step in the Loading Zone (Strategy C). Because the washing step is the major reason for product dilution, one can move the washing step out of the loading zone to a separate washing zone, to achieve a higher product concentration (Strategy C). In this case, one more zone (zone II) is added between the regeneration/reequilibration zone (zone I) and the loading zone (zone III), to keep the protein trailing wave confined in zone II. This strategy results in a three-zone carousel with a zone configuration of 1-1-2, as shown in Figure 4. The operating conditions of this process are determined from the standing wave design (SWD).25,26 VERSE simulations are then conducted to validate the threezone carousel without the washing step. As shown in Figure 5, the protein trailing wave is confined within zone II, whereas the zinc adsorption wave is confined within zone III. These results confirm that high purity and a high yield of protein are maintained throughout the operation of the proposed four-zone carousel process. To achieve even higher protein product concentration than the aforementioned process, one can add one enrichment zone (zone IV) to confine the protein advancing wave (Figure 6). This modification results in a four-zone carousel with a zone configuration of 1-1-2-1, which is shown in Figure 6. The operating conditions of this process are obtained from the SWD.25,26 VERSE simulations are then conducted to validate the four-zone carousel without the washing step. As shown in Figure 7, the trailing and advancing waves of protein are confined within zones II and IV, respectively, while the zinc adsorption wave is confined within zone III. These results confirm that high purity and a high yield of protein are
Ind. Eng. Chem. Res., Vol. 45, No. 3, 2006 1061 Table 2. Operating Conditions of Alternative Chelex Carousel Processesa Strategy A
Strategy B
two-zone carousel with three columns
two-zone carousel with four columns
three-zone carousel without a washing step in the loading zone
four-zone carousel without a washing step in the loading zone
1-2
1-3
1-1-2
1-1-2-1
18.1 12.7 15.3 4.0
12.7 18.1 21.9 4.0
22.5 10.2 12.3 0.69 4.0
22.5 10.2 12.3 0.69 4.0 0.47
4.0 4.0
4.0 4.0
3.31
3.31
28 21 7
40 33 7
4.0 22.5 22.5
3.53 22.5 22.5
zone configuration zone I flow rate (mL/min) regeneration time per cycle (min/cycle) re-equilibration time per cycle (min/cycle) zone II flow rate (mL/min) zone III flow rate (mL/min) zone IV flow rate (mL/min) inlet and outlet flow rates (mL/min) feed wash product switching time (min) loading time (min) washing time (min) a
Strategy C
Column dimension: 1.1 cm ID × 30 cm length. The extra-column dead volume is 1.13 mL per column or 4.0% of the bed volume.
Table 3. Comparison of All the Chelex Carousel Processes Proposed in This Study
total column number throughput per bed volume (L/h/100 L BV) normalized product concentration, Cp/Cf mobile phase consumptionb (L/kg protein) production rateb (kg protein/yr) number of pumps number of valves a
Previous Processa
Strategy A
Strategy B
three-zone carousel with four columns
two-zone carousel with three columns
two-zone carousel with four columns
three-zone carousel without a washing step in the loading zone
four-zone carousel without a washing step in the loading zone
3 210.5 0.75 94.0 126.3 4 3
4 173.6 0.83 59.7 138.7 4 4
4 174.4 0.83 103.7 139.1 4 4
5 138.9 0.94 103.7 139.1 5 5
4 157.8 0.75 94.0 126.3 4 4
Strategy C
Data taken from ref 14. b The concentration of protein in feed was set at 80 g/L.
Figure 4. Schematic of a three-zone Chelex carousel process without washing step in the loading zone (Strategy C, based on three zones).
Figure 3. Two-zone Chelex carousel process with four columns (Strategy B): (a) process diagram, and (b) steady-state column profiles at the end of a switching period from VERSE simulations.
maintained throughout the operation of the proposed four-zone carousel process. An interesting phenomenon occurs in the zinc concentration profile in zone II of the carousel based on the Strategy C. As shown in Figures 5 and 7, the zinc concentration profile in zone II shows a sharp rollup. This phenomenon is caused by the
following two reasons; (1) the abrupt change in the environment of the mobile phase due to port switching and (2) the presence of a sharp gradient of the adsorbed zinc concentration along the axial distance in the column of zone II. 4.4. Comparison of All the Proposed Chelex Carousel Processes. Table 3 compares all the proposed Chelex carousel processes, in terms of the economic factors. Overall, the fourzone carousel based on Strategy C gives the highest product concentration. This is due to less dilution of the product resulting from the addition of enrichment zone and the absence of the washing step in each switching period. However, the four-zone carousel requires more valves and gives lower throughput per bed volume than the other carousel processes, because more columns should be used to contain the protein concentration waves. Compared to the four-zone carousel, the three-zone carousel based on Strategy C has a smaller column number, which leads to fewer valves and higher throughput per bed
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Figure 5. Steady-state column profiles of a three-zone Chelex carousel without washing step in the loading zone (Strategy C, based on three zones): (a) at the beginning of a switching period, (b) in the middle of a switching period, and (c) at the end of a switching period. (The heavy line denotes zinc data, and the thinner line represents protein data.)
Figure 7. Steady-state column profiles of a four-zone Chelex carousel without a washing step in the loading zone (Strategy C, based on four zones): (a) at the beginning of a switching period, (b) in the Middle of a switching period, and (c) at the end of a switching period. (The heavy line denotes zinc data, and the thinner line represents protein data.)
zone carousel, based on Strategy A. Compared to the four-zone carousel based on Strategy C, the two-zone carousel based on Strategy B has fewer valves and a higher throughput per bed volume. Based on these considerations, the two-zone carousel with four columns (zone configuration 1-3) is proposed as the most practical process for the removal of zinc ions from a protein solution. 5. Conclusions
Figure 6. Schematic of a four-zone Chelex carousel process without a washing step in the loading zone (Strategy C, based on four zones).
volume. However, the removal of the protein enrichment zone results in a decrease in product concentration (Table 3). Compared to the carousels based on Strategies A and B, the carousel based on Strategy C has a higher mobile phase consumption (see Table 3). This is due to the additional use of 1 N acetic acid to keep the protein trailing wave confined in zone II (see Figures 5 and 7). The highest throughput per bed volume is obtained by the two-zone carousel based on Strategy A, which also has the fewest valves, because it has the smallest column number. However, the product concentration of this process is considerably lower than the other carousel processes, because of the presence of a long washing step. The two-zone carousel based on Strategy B has the lowest mobile phase consumption. This process also has a higher product concentration and a higher production rate than the two-
Alternative Chelex carousel processes with different zone configurations and zone numbers were developed to improve one or more of the economic factors in the previous three-zone Chelex carousel process with four columns. The highest throughput per bed volume was obtained by combining the regeneration and the re-equilibration zone into one zone (Strategy A). The lowest mobile phase consumption was attained by increasing the length of the loading zone (Strategy B). The highest product concentration was achieved by moving the washing step out of the loading zone into a separate zone (Strategy C). The performances of the proposed carousels based on the aforementioned strategies were verified with results from VERSE simulations. Acknowledgment We are grateful to Dr. Yi Xie from Eli Lilly & Company and Mr. Chim Chin from Purdue University for their valuable inputs and suggestions. We also thank Dr. Fred Larimore from Eli Lilly & Company for inspiring this work.
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Nomenclature Ci ) concentration of component i in the solution phase (equiv/ L) Cf,i ) inlet concentration of component i (equiv/L) Cm,i ) mobile phase concentration of component i (equiv/L) Cp,i ) pore phase concentration of component i (equiv/L) CT ) total solution phase concentration (equiv/L) C h i ) concentration of component i on the resin phase (equiv/L of packed bed) C h T ) total exchange capacity (equiv/L of packed bed) Dp ) intraparticle diffusivity (cm2/min) Eb ) axial dispersion coefficient (cm2/min) Ka ) dissociation equilibrium constant of acetic acid Kf ) film mass-transfer coefficient (cm/min) KZn,H ) mass action equilibrium constant qi ) solid-phase concentration of component i (equiv/L of solid volume) Rp ) particle radius (cm) u0 ) mobile phase interstitial velocity (cm/min) Greek Letters b ) interparticle void fraction p ) intraparticle void fraction Literature Cited (1) Reinhardt, C. G.; Krugh, T. R. A Comparative Study of Ethidium Bromide Complexes with Dinucleotides and DNA: Direct Evidence for Intercalation and Nucleic Acid Sequence Preferences. Biochemistry 1978, 17, 4845. (2) Dunn, M. F.; Pattison, S. E.; Storm, M. C.; Quiel, E. Comparison of the Zinc Binding Domains in the 7S Nerve Growth Factor and the ZincInsulin Hexamer. Biochemistry 1980, 19, 718. (3) Pella, P. A.; Kingston, H. M.; Sieber, J. R. Effect of Sample Dissolution Procedures on X-ray Spectrometric Analysis of Biological Materials. Anal. Chem. 1983, 55, 1193. (4) Putnam-Evans, C. L.; Harmon, A. C.; Cormier, M. J. Purification and Characterization of a Novel Calcium-Dependent Protein Kinase from Soybean. Biochemistry 1990, 29, 2488. (5) Schroeder, S. A.; Roongta, V.; Fu, J. M.; Jones, C. R.; Gorenstein, D. G. Sequence-Dependent Variations in the 31P NMR Spectra and Backbone Torsional Angles of Wild-Type and Mutant Lac Operator Fragments. Biochemistry 1989, 28, 8292. (6) Kinoshita, C. M.; Ying, S.-C.; Hugli, T. E.; Siegel, J. N.; Potempa, L. A.; Jiang, H.; Houghten, R. A.; Gewurz, H. Elucidation of a ProteaseSensitive Site Involved in the Binding of Calcium to C-Reactive Protein. Biochemistry 1989, 28, 9840. (7) Trost, J. T.; Blankenship, R. E. Isolation of a Photoactive Photosynthetic Reaction Center-Core Antenna Complex from Heliobacillus mobilis. Biochemistry 1989, 28, 9898. (8) Vorherr, T.; James, P.; Krebs, J.; Enyedi, A.; McCormick, D. J.; Penniston, J. T.; Carafoli, E. Interaction of Calmodulin with the Calmodulin Binding Domain of the Plasma Membrane Ca2+ Pump. Biochemistry 1990, 29, 355.
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ReceiVed for reView August 20, 2005 ReVised manuscript receiVed November 1, 2005 Accepted November 30, 2005 IE050955W
