Startup and Shutdown Strategies of Simulated Moving Bed for Insulin

Mar 4, 2003 - Jason Bentley , Suzhou Li , and Yoshiaki Kawajiri. Industrial & Engineering Chemistry Research 2014 53 (30), 12063-12076. Abstract | Ful...
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Startup and Shutdown Strategies of Simulated Moving Bed for Insulin Purification Yi Xie, Sung-Yong Mun, and Nien-Hwa Linda Wang* School of Chemical Engineering, Purdue University, West Lafayette, IN 47907-1283

A tandem simulated moving bed (SMB) process, which consists of two SMBs in series, was developed for insulin purification in a previous study (Xie, Y.; Mun, S.-Y.; Kim, J.-H.; Wang, N.-H. L. Biotechnol. Prog. 2002, 18, 1332). A conventional startup procedure required a period of 12 h for the first SMB and 46 h for the second SMB to reach cyclic steady state. In this study, a startup strategy with pre-loading and pre-elution steps has been developed to reduce the startup period to 2 h for the first SMB and 5 h for the second SMB. The transient dynamics of the SMB shutdown process was also studied for the first time. A new shutdown strategy has also been developed to recover more than 99% of the remaining insulin during shutdown. The startup and shutdown strategies are verified with results from computer simulations and validated with experimental data. 1. Introduction A simulated moving bed (SMB) is a continuous separation technique. It has many advantages over conventional batch chromatography, such as high throughput per unit bed volume, low solvent consumption, high purity, and high yield. UOP first introduced SMBs for petrochemical purification in the early 1960s.1 In the following three decades, another major application of SMBs was the production of high-fructose corn syrup (HFCS) in the sugar industry.2,3 Recently, many applications of SMBs for the purification of biochemicals and pharmaceutical products have emerged at various scales.4 In particular, SMBs are widely used for chiral separations.5,6 In a previous study, a tandem SMB (two SMBs in series) process was developed for insulin purification (see Figure 1).7 The mixture to be separated consisted of insulin and other impurities, zinc chloride and highmolecular-weight proteins (HMWP). The HMWP and some zinc chloride were removed from the raffinate port in the first SMB (defined as ring I). Insulin and the rest of the zinc chloride were collected from the extract port and loaded into the second SMB (defined as ring II). In ring II, insulin was recovered from the raffinate port, and zinc chloride was removed from the extract port. As with most of the literature on SMBs, the insulin purification in the previous study was focused on steady state (or cyclic steady state).7 The SMB design methods reported in the literature are all based on steady-state operation.8-14 The advantages of an SMB over batch chromatography are obvious when the SMB is operated at steady state. During the startup period, however, the product concentration and yield are lower than the values at steady state. The low concentration of the product might reduce the efficiency of the following downstream processes. In addition, the long startup period results in low adsorbent productivity. Furthermore, frequent startups and shutdowns might be needed if the feed batches are small and no contamination * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 765-494-4081. Fax: 765-4940805.

Figure 1. Schematic diagram of the tandem SMB process for insulin purification.

between feed batches is allowed. This is especially important in the pharmaceutical industry, where batch identity is critical. Moreover, efficient startup and shutdown strategies can also be applied to SMB processes for large-volume chemical production, where emergency shutdown might occur and normal operation should be resumed quickly to save time and reduce losses of valuable product. Therefore, a short startup period is preferred in SMBs. In the literature, the startup in SMBs is usually referred to as the transient process. However, some researchers also refer to the process between two consecutive switchings as the transient process in SMBs.15 To avoid confusion, we use the term startup

10.1021/ie020674d CCC: $25.00 © 2003 American Chemical Society Published on Web 03/04/2003

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instead of transient process throughout this paper. Studies in the literature have mainly focused on the modeling of SMB startup. Zhong and Guiochon derived analytical solutions for linear systems without masstransfer effects (or ideal systems) to describe the evolution of column profiles during startup.16 Ching et al. used a dispersed plug-flow model to predict the startup processes of SMBs for fructose-dextran and fructoseraffinose separations.17 They treated the SMB as equivalent to a true countercurrent process. A similar approach was adopted by Pais et al. to simulate an SMB process for the separation of 1,1′-bi-2-naphthol enantiomers.18 Later, Pais et al. applied a more rigorous model to account for the actual periodic movement of inlet and outlet ports.19 Meanwhile, Wu et al. used a similar rigorous SMB model to simulate an SMB process for the separation of two amino acids.20 In their study, the effluent histories during the startup period were obtained not only from the extract and raffinate ports, but also from an additional sampling port. The same amino acid binary system was used by Xie et al. to develop a design method based on the impurity breakthroughs during startup.21 The impurity breakthrough time was applied to adjust the SMB operating parameters. Strube et al.22 and Strube and Schmidt-Traub23 also applied a detailed dynamic model for SMBs for chiral separations. They mentioned the importance of modeling in the analysis of startup procedures for SMBs. Zhong et al. studied the effects of the mobile-phase flow rate on the startup process using an equilibrium-dispersive model.24 Yun et al. used the same model to study the effects of column efficiency on the startup process.25 However, none of the aforementioned studies explicitly investigated the optimization of startup procedures. Lim and Ching proposed a pre-loading step so that the SMB operation did not start with clean columns; thus, the startup period was shortened.26 In comparison with the conventional startup, the addition of a preloading step for their chiral separation could reduce the startup period by 1-2 complete cycles. However, the pre-loading step was briefly mentioned without any detailed design procedure, and only the theoretical results were shown. In addition, only the raffinate product was studied. In our previous study of insulin purification, both ring I and ring II were started with clean columns in the conventional way.7 The startup periods were 12 h for ring I and 46 h for ring II. The startup period is defined here as the time required for the product concentration to reach 99% of its cyclic steady-state value. The first of two goals of this study is to develop pre-loading and pre-elution processes to reduce the startup periods for both ring I and ring II. This study will also investigate product recovery during shutdown. No study on the shutdown processes of SMBs has been reported in the literature. Because of the recycle characteristics of SMB, it is difficult to recover the product retained in the columns during the shutdown process. For example, part of the stream (containing the insulin product) from zone I is drawn from the extract port in ring I, but the other part flows into zone II to enrich the product. Theoretically, it would take an infinite number of steps of regular SMB operation to recover all of the product retained in the columns. Obviously, this is not practical. Thus, the

second goal of this study is to design a shutdown process to recover the retained insulin within a reasonably short period. In this study, a batch operation and an SMB operation are implemented alternately to achieve the above goals. The SMB can be easily reconfigured and operated in batch mode during the startup and shutdown processes. Concentration wave analysis and computer simulation are used to search for the optimal strategies for startup and shutdown. The computer simulations show that the startup period can be reduced from 12 h with the conventional startup to 2 h in ring I and from 46 to 5 h in ring II. The simulations also show that 99.8% and 99.4% of the retained insulin can be recovered during the shutdown process for ring I and ring II, respectively. The startup and shutdown strategies are validated experimentally. 2. Wave Analysis and Strategies for Startup and Shutdown 2.1. Wave Analysis. To shorten the startup period, the columns are pre-loaded with the feed so that the insulin column profile at cyclic steady state can be established quickly. This pre-loading step is performed in batch mode at the maximum allowable flow rate, which is limited by the pressure drop over the gel.7 The amount of pre-loaded feed is based on the column profile of insulin at cyclic steady state, which indicates how many columns are saturated with insulin. During the pre-loading step, insulin cannot be separated from the impurities completely. A pre-elution step is needed after the pre-loading step to further separate insulin from the impurities and prevent loss of insulin due to wraparound in the following SMB operation. The pre-elution period is estimated from concentration wave analysis. The concentration wave analysis is based on the local equilibrium theory,27 which relates the mean solute migration velocity (concentration wave velocity) to the interstitial velocity of the mobile phase (u0) and the retention factor (i.e., isotherms, sizeexclusion factors, etc.). The wave velocity (uw) for component i in a size-exclusion system is given by

uw,i )

u0b b + (1 - b)Keip

(1)

where b is the bed voidage, p is the particle porosity, and Kei is the size-exclusion factor of component i. The local equilibrium theory, however, is valid only for ideal systems (i.e., systems in which mass-transfer effects are negligible). The estimation based on the local equilibrium theory is used as an initial guess for the pre-elution duration. Computer simulations that involve a detailed rate model are needed for nonideal systems (i.e., systems in which mass-transfer effects are not negligible) to predict wave spreading due to masstransfer effects. The computer program and intrinsic engineering parameters were validated in our previous study.7 The initially guessed pre-elution period is adjusted on the basis of the simulation results to ensure product purity. A short pre-elution period results in a low-purity extract, but an overly long one results in a low-purity raffinate. After pre-loading and pre-elution, the SMB is operated in the regular SMB mode. Simulations are used to predict the effluent history of insulin at the product port.

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Table 1. Operating Parameters for Regular SMB Operation of Ring I and Ring IIa ring I

ring II

zone configurationb 2-2-4-2 2-3-3-2 feed concentrations (g/L) HMWP 0.023 0 insulin 83.5 69.5 ZnCl2 0.315 0.303 zone flow rates (mL/min) zone I 8.48 8.01 zone II 5.13 6.85 zone III 7.38 7.95 zone IV 5.03 6.66 inlet and outlet flow rates (mL/min) feed 2.25 1.10 eluent 3.46 1.35 raffinate 2.36 1.29 extract 3.35 1.17 switching time (min) 27.4 33.7 a Operating parameters obtained from our previous study7 on the laboratory-scale tandem SMB process for insulin purification. b Zone configuration refers to the distribution of columns among the four zones. For example, 2-4-2-2 means 2 columns in zone I, 4 columns in zone II, 2 columns in zone III, and 2 columns in zone IV.

The simulated effluent history of insulin indicates when the product concentration reaches its cyclic steady-state value. This is used to validate the startup strategy. The shutdown process refers to the process of recovering the insulin remaining in the columns after the feeding is stopped. During the shutdown process, the zone flow rates should be different from those applied during regular operation. Similarly to the design of the startup process, the shutdown process is also designed on the basis of wave analysis and computer simulations. Because the SMB equipment is operated in SMB mode, the net wave velocity, i.e., the difference between the wave velocity (uw) and the bed velocity (ν), is used to estimate the time needed to recover the retained insulin. The effluent history from the simulation is used to validate the estimation. In conventional SMB operation, part of the outflow from zone I is drawn as the extract, and the rest is pumped into zone II. Insulin is recovered at the extract port in ring I. As the shutdown begins, the feed flow is stopped, and the conventional SMB port switching of ring I continues until the insulin front shifts to zone I. At this time, the entire outflow from zone I is collected in the extract stream to recover insulin completely. In ring II, insulin is collected at the raffinate port. During shutdown, the stream to zone IV is stopped, and the entire outflow from zone III is recovered as the raffinate while the port switching continues. More details are given in the next section. 2.2. Strategy for Startup of Ring I. 2.2.1. PreLoading Step. The conventional startup process for insulin purification was simulated using the computer program and the parameters validated in the previous study.7 The operating conditions are listed in Table 1. The effluent histories of ring I and ring II are shown in Figures 2 and 3, respectively. The column profiles at cyclic steady state are shown in Figure 4. The column profile of insulin in ring I at cyclic steady state (Figure 4a) shows that the insulin band is distributed across five columns. One of the five columns contains the front, and another contains the trailing wave. Only three columns are fully saturated; thus, three columns of the insulin feed are loaded during the pre-loading step.

Figure 2. Effluent histories of (a) the extract and (b) the raffinate of ring I under regular SMB operation with the conventional startup procedure.

Figure 3. Effluent histories of (a) the extract and (b) the raffinate of ring II under regular SMB operation with the conventional startup procedure.

To estimate the time needed to saturate three columns with the insulin feed, the concentration wave velocity of insulin (uw,insulin) is calculated as

uw,insulin )

FI/S ) 0.535 (cm/min) b + (1 - b)pKeinsulin (2)

where FI () 8.5 mL/min) is the zone I flow rate of ring I and S is the cross-sectional area of the column. The zone I flow rate, which is the same as the maximum allowable flow rate specified in our previous study,7 is used to minimize the startup time. The pre-loading time

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2.2.2. Pre-Elution Step. After the pre-loading step, the columns are eluted with the eluent through the eluent port (Figure 5b). The column connection remains the same as in the pre-loading step. The length of the pre-elution time determines the impurity level (concentration of HMWP), the insulin concentration level, and the possible loss of insulin in the subsequent SMB operation. Long elution times result in high dilutions of insulin, whereas short elution times result in high HMWP levels remaining at the end of column 2 (the extract port in regular SMB operation mode). If the elution time is too short, some insulin will be lost in the raffinate because the insulin in zone I shifts into zone IV as a result of column switching in the subsequent SMB operation. The pre-elution time is initially estimated on the basis that the insulin concentration wave travels one column length, so that the trailing wave of insulin will leave column 1 and will not cross-contaminate the raffinate. The pre-elution time is given by

Figure 4. Mid-cycle column profiles at cyclic steady state of (a) ring I and (b) ring II.

tpre-elution )

Lc uw,insulin

) 25.6 (min)

(4)

The pre-elution time will be optimized using computer simulations. After the pre-loading and pre-elution steps, the operation mode is switched to conventional SMB mode, starting with the same column order. Table 1 lists the operating parameters of the regular SMB operation. 2.3. Strategy for Startup of Ring II. 2.3.1. PreLoading Step. As for the startup process of ring I, the maximum flow rate of 8.5 mL/min is chosen during the pre-loading step. The associated concentration wave velocity of insulin is 0.535 cm/min according to eq 2. The column profile of insulin at cyclic steady state of ring II (Figure 4b) shows that four columns are completely saturated with insulin. This indicates that four columns should be loaded during the pre-loading step, so the preloading time is given by

tpre-loading )

Figure 5. Column connections during the startup process: (a) pre-loading step in ring I, (b) pre-elution step in ring I, (c) preloading step in ring II, (d) pre-elution step in ring II.

is given by

tpre-loading )

3Lc uw,insulin

) 76.8 (min)

(3)

where Lc is the column length. During the pre-loading step, the feed is loaded through the eluent port, i.e., the inlet of column 1 (Figure 5a). The extract port and the feed port are closed, and the effluent exits at the end of column 6.

4Lc uw,insulin

) 102.4 (min)

(5)

Because of mass-transfer effects, the insulin front might spread into the fifth column during the pre-loading step. For this reason, the feed is loaded into the inlet of column 4, and the effluent exits at the end of column 8 (Figure 5). 2.3.2. Pre-Elution Step. Because of the periodic movement of the inlet and outlet ports of the SMB, the raffinate port always reaches the low end of the insulin front at the beginning of each switching period. To obtain a high concentration of insulin during startup, the insulin front should have passed the raffinate port by the end of the startup process. Therefore, a preelution step is needed to push the insulin front past the raffinate port (exit of column 8 in Figure 5). During the pre-elution step, the eluent is loaded into the inlet of column 4, and the effluent exits at the end of column 9 (Figure 5). Note that insulin is the extract product in ring I but the raffinate product in ring II. Hence, the pre-loading and pre-elution inlet positions of the two rings are different. According to the results of the pre-loading step, the insulin front needs to travel one column beyond the

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raffinate port (exit of column 8) during the pre-elution step. The duration of the pre-elution step is therefore the same as that estimated from eq 4. After the preloading and pre-elution steps, the operation mode is switched to conventional SMB mode (see Table 1 for the operating parameters). 2.4. Strategy for Shutdown of Ring I. The most straightforward strategy for shutdown (case 1) is to replace the feed tank with an eluent tank and to keep the zone flow rates and switching time the same as those of regular operation. Insulin is collected from the extract port, and HMWP is collected from the raffinate port as before. The second strategy (case 2) is based on an analysis of the insulin concentration wave. The insulin front reaches the end of column 7 at cyclic steady state (Figure 4a). To recover insulin in the columns, the insulin front should migrate toward the extract port (exit of column 2) until the insulin front can be separated from the trailing wave of the HMWP. According to the standing wave design7,9 for zone II, HMWP is standing, and insulin is migrating toward the extract port during the conventional SMB operation. During the shutdown process, if the feed flow rate is zero, then the zone III flow rate equals the zone II flow rate. The wave velocity of the insulin front in zone III is lower than the port velocity, and the insulin front shifts toward the extract port. The wave velocity of insulin based on the zone II flow rate is given by

uw,insulin )

FII/S ) 0.323 (cm/min) b + (1 - b)pKeinsulin (6)

where FII () 5.13 mL/min) is the zone II flow rate of ring I. The bed velocity is given by

ν)

Lc ) 0.5 (cm/min) ts

(7)

To shift the insulin front from the end of column 7 to the end of column 3 and to separate it from the trailing wave of HMWP, ring I should be run for the following number of switching steps

4Lc ν - uw,insulin ) 12 (steps) ts

(8)

To prevent possible cross-contamination caused by HMWP from zone IV, the zone IV flow rate is set to zero. As shown in Figure 6a, during the shutdown of ring I, zone III is disconnected from zone IV. The operating parameters of the shutdown process are listed in Table 2 After the insulin front is separated from the trailing wave of the HMWP, ring I is operated in batch mode to recover the rest of the insulin. The entire stream from zone I is collected in the extract. As shown in Figure 6a and b, column 2 is disconnected from column 3 in batch mode. The elution period equals two switching periods so that the insulin retained in columns 1 and 2 can be fully recovered.

Figure 6. Column connection during the shutdown process: (a) SMB operation mode in ring I, (b) batch operation mode in ring I, (c) SMB operation mode in ring II. Table 2. Operating Parameters of the Shutdown Process ring I case 1 zone configuration

case 2

2-2-4-2 2-2-4-2 zone flow rates (mL/min) zone I 8.48 8.48 zone II 5.13 5.13 zone III 7.38 5.13 zone IV 5.03 0 inlet and outlet flow rates (mL/min) feed 2.25 0 eluent 3.46 8.48 raffinate 2.36 5.13 extract 3.35 3.35 switching time (min) 27.4 27.4

ring II 2-3-3-2 8.01 7.95 7.95 0 0 8.01 7.95 0.06 33.7

2.5. Strategy for Shutdown of Ring II. A wave analysis similar to that for ring I can be applied to the design of the shutdown process for ring II. Insulin, however, is collected at the raffinate port in ring II, and the trailing wave of insulin should be analyzed instead. At the cyclic steady state of ring II, the trailing wave of insulin reaches the end of column 3 (Figure 4b). To recover the insulin retained in the columns, the trailing wave of insulin should migrate toward the raffinate port (exit of column 8). According to the standing wave design7,9 for zone III of ring II, zinc chloride is standing, and insulin moves toward the raffinate port during regular SMB operation. During the shutdown process, the feed flow rate is zero, and the flow rate of zone II is increased to equal that of zone III by reducing the extract flow rate. As a result, the velocity of the trailing wave of insulin is higher than the bed velocity, and both the front and the trailing wave of insulin migrate toward the raffinate port. The zone IV flow rate is set to zero (Figure 6c) to prevent loss of insulin from zone IV. The eluent flow rate is increased to maintain the zone I flow rate.

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The wave velocity of insulin based on the zone III flow rate is given by

uw,insulin )

FIII/S ) 0.500 (cm/min) b + (1 - b)pKeinsulin (9)

where FIII () 7.95 mL/min) is the zone III flow rate of ring II. The port velocity is given by

ν)

Lc ) 0.407 (cm/min) ts

(10)

To allow the trailing wave of insulin to shift from the end of column 3 to the end of column 8 and to separate it from the zinc chloride front, ring II shutdown should be run for the following number of steps

5Lc uw,insulin - ν ) 22 (steps) ts

(11)

See Table 2 for the other operating parameters of the shutdown process. After 22 steps of SMB operation, ring II does not need to operate in batch mode as adopted in the ring I shutdown. In ring I, part of the zone I stream flows into zone II during the 12 steps of shutdown in SMB mode. Some insulin is retained in the column downstream from the extract port. The batch operation is needed to recover this part of the insulin. In ring II, however, the entire zone III stream is collected as the product, and the column downstream from the raffinate port does not contain insulin; thus, batch operation is not needed. 3. Experimental Section 3.1. Materials and Equipment. Insulin powder containing zinc chloride (0.4 wt %) and HMWP (0.02 wt %) was dissolved in 1 N acetic acid and used as the feed for the SMB experiment. Glacial acetic acid was purchased from Mallinckrodt Baker Inc. (Paris, KY). Distilled deionized water (DDW) was obtained from a Milli-Q system manufactured by Millipore (Bedford, MA). The separation medium was Sephadex gel with an average radius of 54 µm. All of the columns and fittings were purchased from Ace Glass Inc. (Louisville, KY). The columns had a diameter of 5.10 cm and a packing length of 13.7 cm. A Vydac C-18 HPLC column (25 cm × 4.6 mm) for protein and peptide analysis was purchased from Vydac Co. (Hesperia, CA) and used for the insulin assay. A Waters insulin/HMWP size-exclusion HPLC column (30 cm × 7.8 mm) was purchased from Millipore Corp. (Milford, MA) and used for the HMWP assay. The HPLC system consists of two pumps (Waters 510), a tunable single-wavelength detector (Waters 486), and an injector (Waters U6K). Waters Millennium 2010 software running in the Windows environment was used for data collection and analysis. A laboratory-scale SMB unit was purchased from Advanced Separation Technology (AST, Lakeland, FL). It consists of a controller for the adjustment of the switching time and a frame that supports a rotation gear, a drive assembly, and a column rack. The unit can be easily switched between SMB and batch operation by changing the tubing connections from the central

rotation valve to the columns. The flow rates of the feed and eluent were controlled by two FPLC pumps (P-500) purchased from Pharmacia (Piscataway, NJ). Two singlepiston pumps (model RHV) purchased from Fluid Metering Inc. (Syosset, NY) were used to control the flow rates for zones II and IV. 3.2. Procedure. During the startup process, the AST unit was operated in batch mode. The column connections are shown in Figure 5a. The feed was loaded into column 1. The flow rate was 8.48 mL/min, and the loading period was 76.9 min. The pre-loading step was followed by a 37-min elution with 1 N acetic acid, which was also loaded into column 1 (Figure 5b). The elution flow rate was 8.48 mL/min. After the startup process, the AST unit was switched to SMB mode and operated under the conditions listed in Table 1 (ring I). The flow rates and switching time were the same as those in our previous study.7 However, the feed composition in the experiments of this study was different from that provided in Table 1. The feed consisted of 73.0 g/L insulin, 0.284 g/L zinc chloride, and 0.013 g/L HMWP. Because this process involves sizeexclusion chromatography and the system is linear, the flow rates and switching time are independent of the feed concentrations. After 210 steps of SMB operation, the feed pump was turned off, and the shutdown process was started. During the shutdown period, the AST unit was first operated in SMB mode for 12 steps and then operated in batch mode for 54.8 min at a flow rate of 8.48 mL/ min. The shutdown operating conditions are listed in Table 2 (ring I, case 2), and the column connections are shown in Figure 6. 4. Results and Discussion 4.1. Startup of Ring I (Simulations). 4.1.1. PreLoading Step. As expected, the insulin band spans three columns (Figure 7a) after pre-loading. Because of mass-transfer effects, the insulin front extends into column 4. The HMWP front migrates faster than the insulin front. Partial separation is achieved. Note that the feed composition listed in Table 1 was used in the simulations. 4.1.2. Pre-Elution Step. Case 1. According to the design procedure, one of the goals of pre-elution is to remove insulin from column 1 to prevent loss of insulin to the raffinate. Figure 7b shows that this goal is fulfilled. However, in this case, the concentration of HMWP (CHMWP) at the extract port (column 2) by the end of the pre-elution step is 23.0 mg/L, whereas the concentration of insulin (Cinsulin) at the same position at the same time is 83.5 g/L. This gives

CHMWP ) 275 (ppm) Cinsulin

(12)

The impurity level is much higher than allowed. Therefore, additional elution time is needed. Case 2. To estimate the additional elution time, the wave velocity of the HMWP (uw,HMWP) is calculated

uw,HMWP )

u0b b + (1 - b)pKeHMWP

) 0.904 (cm/min) (13)

As indicated from the HMWP column profile of case 1

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Figure 8. Effluent histories of (a) the extract and (b) the raffinate of ring I under regular SMB operation with the pre-loading and pre-elution steps (case 1).

Figure 7. Column profiles of the startup process of ring I: (a) pre-loading, (b) pre-elution (case 1), (c) pre-elution (case 2).

(Figure 7b), the trailing wave of HMWP needs to travel an additional distance of 0.75Lc to reach an acceptable concentration level. Then, the total pre-elution time in case 2 is given by

tpre-elution )

Lc uw,insulin

+

0.75Lc ) 37.0 (min) uw,HMWP

(14)

As shown in Figure 7c, after 37.0 min of elution, the HMWP level is reduced significantly. In both cases, no insulin is observed at the exit (the end of column 6). In other words, there is no loss of insulin during the pre-loading and pre-elution steps. 4.1.3. Conventional SMB Operation of Ring I after Pre-Loading and Pre-Elution. Case 1. Starting from the first step, Cinsulin in the extract is higher than the value at cyclic steady state (Figure 8a). However, Cinsulin is lower than the value at cyclic steady state during the period from 400 to 800 min. Furthermore, the HMWP concentration level is higher than allowed, and the effluent composition is unacceptable. This is consistent with the above discussion on the pre-elution of case 1. Case 2. In this case, the HMWP concentration level satisfies requirements. At the first step, Cinsulin in the extract is slightly lower than the value at cyclic steady state (Figure 9a). At the beginning of the second step, Cinsulin in the extract is higher than the value at cyclic steady state. Compared with Figure 2, the effluent history of insulin at the extract port in Figure 9 shows that the

Figure 9. Effluent histories of (a) the extract and (b) the raffinate of ring I under regular SMB operation with the pre-loading and pre-elution steps (case 2). Table 3. Comparison of the Period of Low Product Concentration in the SMB Process with and without the Pre-Loading and Pre-Elution Steps period of low product concentration (h) without pre-loading and pre-elution steps with pre-loading and pre-elution steps

ring I

ring II

12

46

2

5

period of low-concentration product is significantly reduced. This period is reduced from 12 to 2 h (Table 3). 4.2. Startup of Ring II (Simulations). 4.2.1. PreLoading Step. Four columns were saturated with insulin after the pre-loading step, as designed (Figure

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Figure 10. Column profiles of the startup process of ring II: (a) pre-loading, (b) pre-elution.

10a). The insulin front spreads into column 8 but does not reach the end of column 8 by the end of the preloading step (Figure 10a). The insulin front is partially separated from the zinc chloride front. 4.2.2. Pre-Elution Step. By the end of the preelution step, the insulin front crosses the end of column 8 but does not reach the end of column 9 (Figure 10b). Meanwhile, the zinc chloride front does not reach the end of column 8, so it will not contaminate the raffinate during subsequent SMB operation. After the pre-loading and pre-elution steps, the column profile of insulin is similar to that at cyclic steady state (Figure 4b). 4.2.3. Conventional SMB Operation of Ring II after Pre-Loading and Pre-Elution. The effluent history of insulin at the raffinate port is predicted by simulation and shown in Figure 11b. Starting from the first step, the insulin concentration in the raffinate is close to its value at cyclic steady state. In comparison, if no pre-loading and pre-elution steps are applied, 83 switching steps (about 46 h) are required for the insulin concentration in the raffinate to reach 99% of its value at cyclic steady state (Figure 3). In other words, the startup period during which the insulin concentration in the raffinate is lower than 99% of its value at cyclic steady state is shortened from 46 h to about 5 h (Table 3). 4.3. Shutdown of Ring I (Simulations). The amount of insulin retained in the columns of ring I at cyclic steady state is estimated by subtracting the accumulated amount of insulin in both the extract and the raffinate from the total amount of insulin loaded into ring I. This estimation is based on the results of the simulation with the operating parameters listed in Table 1. The estimated amount of insulin retained in the columns is 41.4 g. The total bed volume of the columns is 2.8 L. In case 1, 40.9 g (i.e., 98.9%) of insulin is recovered after 45 steps of SMB operation. Figure 12 shows the dynamics of the column profiles, and Figure 13 shows the effluent histories during the shutdown process of case 1. Because the zone flow rates and switching time in the shutdown process of case 1 are the same as those

Figure 11. Effluent histories of (a) the extract and (b) the raffinate of ring II under regular SMB operation with the preloading and pre-elution steps.

Figure 12. Column profiles of the shutdown process of ring I at (a) the 4th step, (b) the 8th step, and (c) the 12th step (case 1).

during conventional operation, the insulin front is standing in zone III according to the standing wave design. As shown in Figure 12, the insulin in zone III is diluted with time, but the front does not move toward the extract port. As a result, the concentration of insulin

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Figure 13. Effluent histories of (a) the extract and (b) the raffinate of ring I during the shutdown process (case 1).

Figure 15. Effluent histories of (a) the extract and (b) the raffinate of ring I during the shutdown process (case 2). Table 4. Comparison of Two Cases of the Shutdown Process for Ring I average Cinsulin recovery duration of CHMWP/Cinsulin in extract (g/L) (%) shutdown (h) in extract (ppm) case 1 case 2

Figure 14. Column profiles of the shutdown process of ring I at (a) the 4th step, (b) the 8th step, and (c) the 12th step (case 2).

in the extract decreases within a short period, but it takes a long time to recover the remaining 1.1% of insulin. Figure 14 shows the dynamics of the column profile during the shutdown process of case 2. Because the flow rate of zone III is reduced to that of zone II, the velocity

9.91 26.3

98.9 99.8

21 (45 steps) 7 (14 steps)

6.83 1.10

of the insulin front is lower than the port velocity; thus, the insulin front shifts toward the extract port. By the end of 12 switching steps, the insulin front is separated from the trailing wave of HMWP (Figure 14), and the insulin peak is confined within columns 2 and 3. Because of the recycle effects of SMB operation, more time will be required to recover the insulin in columns 2 and 3 under conventional SMB operation. Two switching periods of batch elution are then used to recover the insulin held in columns 2 and 3. By the end of 14 steps in the shutdown process, 41.3 g (i.e., 99.8%) of insulin is recovered, and the concentration of insulin at the extract port is close to zero (Figure 15). As shown in Table 4, the product is more diluted in case 1 than in case 2. In addition, the impurity in case 1 is much higher than that in case 2. 4.4. Shutdown of Ring II (Simulations). The amount of insulin retained in the columns of ring II at cyclic steady state is estimated following the same procedure as used for ring I. The estimated amount of insulin retained in the columns of ring II at cyclic steady state is 61.8 g. The number and dimensions of the columns in ring II are the same as those in ring I. By the end of 22 switching steps (12.4 h) of the shutdown process, 61.4 g (i.e., 99.4%) of insulin can be recovered. The average product concentration during the shutdown process is 10.4 g/L. The average zinc chloride concentration is 4.13 × 10-5 g/(g of insulin), which satisfies the product requirements. The dynamics of the column profiles and the effluent histories during the shutdown process are shown in Figures 16 and 17, respectively. The startup and shutdown procedures for both ring I and ring II are summarized in Tables 5 and 6, respectively. Note that the shutdown period of ring II is much

Ind. Eng. Chem. Res., Vol. 42, No. 7, 2003 1423 Table 5. Summary of the Startup Procedure for the Tandem SMB ring I

ring II

pre-loading step duration (min) 76.9 feed inlet column 1 effluent outlet column 6 flow rate (mL/min) 8.50

102.4 column 4 column 8 8.50

pre-elution step 37.0 column 1 column 6 8.50

25.6 column 4 column 9 8.50

duration (min) eluent inlet effluent outlet flow rate (mL/min)

Table 6. Summary of the Shutdown Procedure for the Tandem SMB duration (min) SMB modea batch mode eluent inlet in batch mode effluent outlet in batch mode

ring I

ring II

329 (12 steps) 54.8 column 1 column 2

742 (22 steps) -

a See Table 2 for the operating parameters for ring II and case 2 in ring I.

Figure 16. Column profiles of the shutdown process of ring II at (a) the 1st step, (b) the 11th step, and (c) the 22nd step.

Figure 17. Effluent histories of (a) the extract and (b) the raffinate of ring II during the shutdown process.

longer than that of ring I. This is mainly due to the different selectivities of the two rings. Insulin is separated from HMWP in ring I and from zinc chloride in ring II. The selectivity of insulin/HMWP is larger than that of insulin/zinc chloride.7 Low selectivity results in a long separation time and, thus, a long recovering

period during shutdown. The startup and shutdown procedures are designed for a laboratory-scale tandem SMB process in this study, but the same design approach can be applied to a pilot-scale or production-scale SMB process. 4.5. Experimental Validation. As discussed in the previous study, the separation between insulin and HMWP was crucial. Ring I was more important than ring II. Therefore, an experiment was performed to validate the startup and shutdown processes for ring I only. The effluent history of insulin at the extract port is shown in Figure 18a. The experimental data are in close agreement with the simulation results. The concentration of insulin in the first step of regular SMB operation is close to its value at cyclic steady state. During the following eight steps, the insulin concentration is even higher than its value at cyclic steady state. As a result, the period of low product concentration is confined within the startup period, which is about 2 h. During the startup process, samples were taken from the exit of column 6 (Figure 5a and b). No insulin was found in the samples. Using the average concentration of the extract and the total amount of loaded insulin, the overall yield over 210 steps in the regular SMB operation was 95.7%. This value was the same, within experimental accuracy, as the value predicted from the computer simulation (96.0%). The raffinate sample analyses showed that less than 0.3% of the insulin was lost at the raffinate port. Both experimental and simulation results indicated that about 4% of the total loaded insulin remained in the columns at the end of the 210th step. Figure 18c shows the effluent histories at the extract port during the shutdown process. Samples from the 12 steps of the shutdown SMB operation were mixed and analyzed. The mixture concentration was used to calculate the yield during these 12 steps of operation. The yield was 3.5%, based on the total amount of the loaded insulin. The concentration of HMWP in the extract during the shutdown process was below the assay limit and close to zero. The following batch operation of the shutdown process recovered 0.2% of the loaded insulin. When the recovery of insulin during the batch operation of shutdown is added to the yields of the regular SMB

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Acknowledgment The authors highly appreciate the help of Mr. Chim Chin and Mr. Ho-Joon Lee in the sample collection and assays. The cold room used for the experiments was provided gratis by the Food Science Department at Purdue University with the permission of Prof. Philip Nelson. We also acknowledge the financial support of grants from NSF (0215146) and the Indiana 21st Century Research and Technology Fund. Literature Cited

Figure 18. Experimental validation of the startup and shutdown procedures of SMB ring I for insulin purification: (a) extract effluent history during startup, (b) raffinate effluent history during startup, (c) extract effluent history during shutdown. exp., experiment; sim., simulation.

operation and the shutdown SMB operation, one can obtain 99.4% yield for the entire process. The experimental yield is in close agreement with the simulation prediction (99.7%). 5. Conclusions A startup procedure has been developed for insulin purification using a tandem SMB. Compared to the conventional startup procedure, the new startup procedure reduces the startup period from 12 to 2 h in ring I and from 46 to 5 h in ring II. The tandem SMB operation with the proposed startup procedure satisfies the purity requirement and achieves a high yield of 99.0%. A shutdown procedure has also been developed for the tandem SMB for insulin purification. The procedure requires 7 h in ring I and 12 h in ring II to recover 99.8% and 99.4% of the insulin remaining in the columns, respectively. The impurity levels in both the ring I and ring II product streams during the shutdown process are below the maximum allowable values. The design method, which uses concentration wave analysis and numerical simulations to develop the startup and shutdown processes, can be generalized to other SMB processes for linear isotherm or size-exclusion systems. The efficient startup and shutdown procedures enable the SMB to carry out frequent small batch purifications.

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Ind. Eng. Chem. Res., Vol. 42, No. 7, 2003 1425 (20) Wu, D.-J.; Xie, Y.; Ma, Z.; Wang, N.-H. L. Design of Simulated Moving Bed Chromatography for Amino Acid Separations. Ind. Eng. Chem. Res. 1998, 37, 4023. (21) Xie, Y.; Wu, D.-J.; Ma, Z.; Wang, N.-H. L. Extended Standing Wave Design Method for Simulated Moving Bed Chromatography: Linear Systems. Ind. Eng. Chem. Res. 2000, 39, 1993. (22) Strube, J.; Altenho¨ner, U.; Meurer, M. Schmidt-Traub, H.; Schulte, M. Dynamic Simulation of Simulated Moving-Bed Chromatographic Processes for the Optimization of Chiral Separations. J. Chromatogr. A. 1997, 769, 81. (23) Strube, J.; Schmidt-Traub, H. Dynamic Simulation of Simulated-Moving-Bed Chromatographic Processes. Comput. Chem. Eng. 1998, 22, 1309. (24) Zhong, G.-M.; Smith, M.-S.; Guiochon, G. Effect of the Flow Rates in Linear, Ideal, Simulated Moving-Bed Chromatography. AIChE J. 1997, 43, 2960.

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Received for review August 28, 2002 Revised manuscript received January 20, 2003 Accepted January 20, 2003 IE020674D