Ind. Eng. Chem. Res. 2003, 42, 4055-4067
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Separation of FTC-Ester Enantiomers Using a Simulated Moving Bed Yi Xie, Benjamin Hritzko,† Chim Yong Chin, and Nien-Hwa Linda Wang* School of Chemical Engineering, Purdue University, Forney Hall, 480 Stadium Mall Drive, West Lafayette, Indiana 47907-2100
A simulated moving bed (SMB) process has been developed for the resolution of racemic mixtures of cis-(()-FTC-ester, a precursor of a potential anti-HIV drug. Chiralpak AD was chosen as the stationary phase and methanol as the mobile phase for this study. The nonlinear standing wave design method was used to determine the zone flow rates and step times of the SMB process. Computer simulations and several laboratory-scale SMB experiments were conducted to test the proposed SMB processes. Extracolumn dead volume (DV) had significant effects on the performance of the laboratory-scale SMB with small columns (10 × 1 cm). Nonuniformly distributed DV was considered in the design and simulations. Different pump arrangements were investigated to reduce the DV effects. High purity (99.8%, or 99.6% e.e.) and high yield (98.9%) were achieved in the SMB experiments. If mass-transfer effects were ignored in the design, the yield would have been lower than 93%. 1. Introduction FTC [cis-(-)-2′-deoxy-5-fluoro-3′-thiacytidine], also known as emtricitabine, is an inhibitor of reverse transcriptase in retroviruses. It has been shown to be potent, nontoxic, and selective against HIV-1 and HIV-2.1,2 Preliminary clinical trials indicate that FTC is 6-10 times stronger against HIV-1 than a competitor, 3TC [cis-(-)-2′,3′-dideoxy-3′-thiacytidine],3 which is sold as Epivir (lamivudine) by GlaxoSmithKline. FTC can be produced through a semisynthetic reaction sequence.4 The reaction sequence can be configured to arrive at an ester (Figure 1a) or an alcohol (Figure 1b), both of which exist as racemates. The desired product is the alcohol (FTC, MW ) 247), but the ester (FTC-ester, MW ) 317) can be easily hydrolyzed to yield the alcohol. Because the cis-(-) form is more active than the cis-(+) form, the desired product is a single enantiomer in the cis-(-) form with a purity of 99.0% or higher. A chromatography step can be employed to separate the desired enantiomer from the racemic mixture. When chromatography is employed at the pilot scale to produce kilogram quantities or at the process scale to produce tons, it is often advantageous in terms of yield, solvent consumption, and sorbent productivity to use simulated moving bed (SMB) technology.5 SMB was originally developed at UOP for petrochemical separations.6 A standard four-zone SMB unit (Figure 2) has ports for the introduction of a feed solution and an eluent (or desorbent), as well as two outlet ports, one for the recovery of the compound with a higher affinity for the stationary phase (the extract) and one for the recovery of the compound with a lower affinity (the raffinate). The inlet and outlet ports move periodically in the direction of the fluid flow, such that the feed is always loaded into the overlapping region of the two * To whom correspondence should be addressed. E-mail:
[email protected]. Tel.: 765-494-4081. Fax: 765-4940805. † Current address: Pfizer, Inc., Groton, CT 06340.
Figure 1. Molecular structures of (a) cis-(()-2′,3′-dideoxy-3-fluoro5′-thiacytidine butyrate [(()-FTC-butyrate or -ester] and (b) cis(()-2′,3′-dideoxy-3-fluoro-5′-thiacytidine [(()-FTC].
Figure 2. Schematic diagram of a four-zone simulated moving bed (SMB). (a) Step N, (b) step N + 1. b, Low-affinity component; 2, high-affinity component.
components whereas the extract (the pure high-affinity component) and the raffinate (the pure low-affinity
10.1021/ie030225t CCC: $25.00 © 2003 American Chemical Society Published on Web 07/29/2003
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component) are always drawn from the separated regions. Compared to conventional batch chromatography processes, SMB gives higher throughput per bed volume, lower solvent consumption, higher yield, and higher purity. A large number of SMB processes have been applied to the separation of petrochemicals and the preparation of high-fructose corn syrup.7 Since the 1990s, SMB processes have been used for chiral separations. The first example of chiral separations with SMBs was given by Negawa and Shoji,8 where Chiralcel OD was used to separate 1-phenylethanol enantiomers. Compared with batch chromatography, the SMB process increased productivity by 60-fold and decreased eluent consumption by 86-fold. The productivity comparison between batch chromatography and SMB is, however, casedependent. In a recent study on the chiral separation of a precursor of a cardiotonic drug, Jupke et al. found that the productivity of the optimized SMB process was about 25% higher than that of the optimized batch chromatography process.9 They attributed such a small difference between the two processes to the low solubility of the product. Many different chiral separations using SMBs have occurred since 1992. Juza et al.10 and Schulte et al.11 reviewed these applications. Many pharmaceutical companies realize the efficiency and low cost of SMB processes for chiral separations. For example, UCB Pharma of Belgium has been using SMBs to produce multi-tons of pure enantiomers since 1998.12 To date, the largest SMB (with columns of 800-mm i.d.) for chiral separations has been installed at Aerojet Fine Chemicals (Sacramento, CA) by Novasep (Vandoeuvre-le`sNancy, France). The major goal of this project is to develop and validate an SMB process for the resolution of FTC-ester enantiomers. Solvent screening experiments were carried out first, followed by estimations of isotherm parameters and intraparticle diffusivities (defined here as the intrinsic engineering parameters) for the most promising chiral stationary phase (CSP)/solvent pair. FTC-ester was more soluble than FTC in all of the tested solvents, indicating that it would be more economical to produce FTC-ester and then convert the ester to FTC. Methanol with Chiralpak AD was found to be the best solvent/sorbent pair for the separation. A series of pulse tests with different loadings of the racemic FTCester at three different flow rates gave estimated isotherm parameters. The isotherm parameters were fine-tuned later using the frontals of optically pure enantiomers and SMB data. Mass-transfer parameters were estimated via HETP analysis. The standing wave design for nonlinear systems was developed to find zone flow rates and step times (defined here as the design parameters) from the intrinsic engineering parameters. Extracolumn dead volume (DV) was found to be critical to the performance of the laboratory-scale SMB unit with small columns. The effects of nonuniformly distributed DV resulting from an internal recycle pump were considered in the design and simulations. Three SMB experiments were carried out. A product purity of 99.6% or higher was achieved in all the experiments. The highest product yield was 98.9%, which was achieved by placing the recycle pump between zone I and zone IV. The results of this study indicate that the standing wave design method can ensure high purity and high yield for nonlinear binary separations.
2. Theory 2.1. Estimation of Isotherm and Mass-Transfer Parameters from Racemic Mixtures and Pure Standards. 2.1.1. Isotherm Parameters. Isotherms are the critical parameters needed to design a chromatography or SMB process. Among the many isotherm models used to correlate parameters, the competitive Langmuir isotherm model (eq 1) is the simplest and the most widely used.
qi )
aiCi N
1+
(i ) 1, 2, ..., N)
(1)
bjCj ∑ j)1
The shock wave velocity of a pure component with a Langmuir isotherm can be described by the equation
L tr,sh
) us )
u0b
|
∆q b + (1 - b)p + (1 - b)(1 - p) ∆C C)Cp (2a)
where
|
∆q a ) ∆C C)Cp 1 + bCp
(2b)
tr,sh is the shock wave retention time, and Cp is the plateau concentration. The Langmuir isotherm a and b parameters can be estimated from a series of frontal tests at different plateau concentrations. The isotherms measured from pure-component frontals can be validated with mixture frontals. The competitive Langmuir model applies only to monolayer adsorption of noninteracting solutes on a surface lattice. This model is thermodynamically consistent only when all solutes have the same saturation capacity (a/b ratio).13 It is well-known that this model cannot accurately describe the adsorption isotherms of some chiral systems because the assumptions of the model are invalid.14 For such systems, one would need to use different Langmuir parameters to explain the chromatograms at different concentrations. Because of its simplicity, the Langmuir model has been used successfully for the design and analysis of many chromatography and SMB systems.13-15 2.1.2. Mass-Transfer Parameters. Mass-transfer parameters are used in the simulation and design of both batch chromatography and SMB processes. The mass-transfer parameters needed are the axial dispersion coefficient (Eb), film mass-transfer coefficient (kf), Brownian diffusivity (D∞), and intraparticle diffusivity (Dp). The film mass-transfer coefficient can be estimated from the Wilson and Geankoplis correlation.16 The Brownian diffusivity can be calculated from the Wilke and Chang correlation.17 The axial dispersion coefficient and the intraparticle diffusivity can be estimated from the low-loading pulse tests using the HETP equation13 as follows
( )( )(
Eb Rp2 u0 b Rp σ2 HETP ) + ) + 2L L 1 - b 3kf 15pDp 2(t )2 u0L r
)
(3) where σ2 ) A/(2πh), A is the peak area, h is the peak
Ind. Eng. Chem. Res., Vol. 42, No. 17, 2003 4057
height, and Rp is the particle radius. Once the isotherm and mass-transfer parameters are known, the chromatography processes can be simulated. 2.2. Rate Model for Computer Simulations. The VERSE simulation package was developed at the Bioseparation Laboratory of Purdue University in the 1980s and is still being elaborated. VERSE is based on a detailed rate model for both the mobile phase and the stationary phase. The rate model consists of the partial differential equations for unsteady-state multicomponent convection, dispersion, and film diffusion in the mobile phase and diffusion in the stationary phase. The equations and associated boundary and initial conditions are discretized by orthogonal collocation on finite elements. The resulting ODEs and algebraic equations are solved using an equation solver. The model equations and the numerical solution procedure can be found in the literature.18,19 The VERSE package has been validated with many different chromatography, carousel, and SMB applications.18-25 If the adsorption isotherms and mass-transfer parameters are accurate and the assumptions of the rate model are valid, VERSE simulations can give reliable predictions of column profiles, effluent histories, and product purities and yields. 2.3. Design of the SMB Process. One of the key issues in developing SMB processes is the determination of the zone flow rates and switching time. Many literature methods for SMB design have been reported, such as the safety margin method,26,27 the triangle theory,28-30 and the standing wave design.31,32 The safety margin method and the triangle theory were developed in the framework of equilibrium theory for ideal systems (without mass-transfer effects). For nonideal systems (defined here as systems with significant mass-transfer effects), an empirical factor is needed in the safety margin method to counter the wave spreading that occurs as a result of mass-transfer effects. In the triangle design method for nonideal systems, a series of distorted triangular regions are generated by many computer simulations to include an infinite number of possible operating conditions.30,33 The standing wave design, however, provides unique solutions for ideal and nonideal systems to guarantee high purity and high yield. The standing wave design (SWD) approach has been successfully used to design SMB processes for ideal and nonideal systems with linear isotherms.31,34-36 The SWD has also been extended to ideal systems with nonlinear isotherms.32 The SMB processes in this study, however, involve nonideal systems with nonlinear isotherms (Langmuir isotherms). The SWD must therefore be modified for the design of nonlinear nonideal SMB processes. Note that we use Langmuir isotherms as the nonlinear isotherms in this study. The flow rate correction terms for overcoming masstransfer spreading in linear systems are assumed to be applicable to nonlinear systems. The trailing parts of the diffuse waves of nonlinear systems (in zones I and II) are similar to the waves in linear systems. The masstransfer correction terms for the flow rates of zones I and II in linear systems can be directly applied to nonlinear systems. The shock waves of nonlinear systems (in zones III and IV) are self-sharpening. The mass-transfer corrections terms for the flow rates of zones III and IV in linear systems are overestimated for nonlinear systems. Therefore, this design approach
is conservative. The SWD equations for nonlinear nonideal systems in this study are as follows
1-
uI0 (1 + PδI2)ν -
1-
(1 +
]
) -∆I (4a)
]
) -∆II (4b)
Pν2(δI2)2 kI2
)
(1 + PδII 1 )ν
βII 1 II (1 + PδII 1 )νL
uIII 0 (1 + PδIII 2 )ν (1 +
EIb2 +
PδI2)νLI
PδIII 2 )νL
uIV 0 (1 + PδIV 1 )ν
[
EII b1 +
2 Pν2(δII 1)
kII 1
) βIII 2
1-
[
βI2
uII 0
-
1-
)
[
EIII b2 III
+
]
) ∆III (4c)
]
) ∆IV (4d)
2 Pν2(δIII 2 )
kIII 2
) βIV 1
[
IV (1 + PδIV 1 )νL
EIV b1 +
2 Pν2(δIV 1 )
kIV 1
δI2 ) p + (1 - p)a2
(4e)
a1 δII 1 ) p + (1 - p) 1 + b2Cp2
(4f)
a2 δIII 2 ) p + (1 - p) 1 + b1Cs1 + b2Cs2
(4g)
a1 δIV 1 ) p + (1 - p) 1 + b1Cp1
(4h)
FF II ) uIII 0 - u0 bS
(4i)
where the subscripts 1 and 2 stand for the low-affinity solute and the high-affinity solute, respectively; the subscripts F, R, D, and E stand for feed, raffinate, desorbent (eluent), and extract, respectively; the superscripts I, II, III, and IV stand for the four zones; ν is the average port velocity () column length/switching time); u0 is the interstitial velocity; b is the interparticle void fraction; p is the intraparticle void fraction; P is the phase ratio, defined as (1 - b)/b; S is the crosssectional area of the column; L is the zone length; a and b are Langmuir isotherm parameters; Eb is the axial dispersion coefficient; F is the volumetric flow rate; and k is a lumped mass-transfer parameter that is defined for each solute i as
Rp2 Rp 1 ) + (i ) 1, 2) ki 15KeipDpi 3kfi
(5)
where Rp is the radius of a resin particle, Dp is the intraparticle diffusivity, kf is the film mass-transfer
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Figure 3. Schematic SMB column profiles of a binary system with nonlinear isotherms.
coefficient, and Ke is the size exclusion factor. β is the logarithm of the ratio of the highest concentration to the lowest concentration of a standing wave in a particular zone. β is an index of product purity and yield; the larger the β value, the higher the product purity and yield.31,36 The Cp and Cs parameters in eq 4 represent plateau concentrations, as shown in Figure 3. An iteration procedure developed by Xie et al.37 can be used to solve eq 4. Equation 4 is derived without considering any extracolumn dead volume effects. Extracolumn dead volume causes additional wave spreading and a delay in wave migration. The delay results in an apparent increase in retention time. To account for the delay, an apparent retention factor δ* is derived to include the retention time in the extracolumn dead volume. δ* is given by
δ* ) δ +
DV PLcSb
(6)
where Lc is the single column length. The apparent retention factor δ* can be substituted into eq 4 to obtain the zone flow rates and switching time with which the SMB process can counter the delay caused by the extracolumn dead volume. 3. Experimental Section 3.1. Materials and Equipment. Racemic FTC-ester was provided by Abbott Laboratories (Chicago, IL). HPLC-grade methanol, which was used as the mobile phase and the solvent for dissolving FTC-ester, was purchased from Mallinckrodt Baker, Inc. (Paris, KY). A tracer, 1,3,5-tri-tert-butylbenzene, which was used to check the bed void fraction, was purchased from Aldrich Chemical Co. (Milwaukee, WI). An analytical column (ChiralPak AD, 25 × 0.46 cm) for the assay of FTC-ester and eight semipreparative columns (ChiralPak AD, 10 × 1 cm) for SMB were purchased from Chiral Technologies, Inc. (Exton, PA). The particle sizes of the analytical column and the semipreparative columns were 10 and 27 µm, respectively. A Waters HPLC system (Milford, MA) was used for the FTC-ester assay and batch chromatography experiments. This HPLC system included two 515 HPLC pumps, a Rheodyne 7725i injector, and a PDA detector. Millennium software was used to control the HPLC. A laboratory-scale versatile SMB unit was constructed in our laboratory. The details of the SMB unit have been
Figure 4. Arrangement of columns and pumps in the laboratoryscale SMB unit.
reported elsewhere.38 For the versatile SMB to function as a four-zone SMB in this project, eight rotary valves and eight columns were used. The number of columns in each zone can be varied by changing the connection paths between the columns and the rotary valves. A computer with LabView controlled valve switching. Four pumps were used to control the flow rates (Figure 4). Two FPLC pumps (Pharmacia, Piscataway, NJ) were used to control the feed and the desorbent (eluant) flow rates. The other two pumps were dual-piston HPLC pumps (Waters), which were used to control the raffinate flow rate and the zone II flow rate in the first two SMB runs. The zone II pump along with the other three pumps controlled the other three zone flow rates. The extract flow rate was the difference in flow rate between zones I and II. In the third SMB run, the desorbent pump was used to control the zone I flow rate. The zone II pump was moved to draw the extract. 3.2. Procedure. Assay. The concentration of FTCester was analyzed by HPLC using the analytical Chiralpak AD column. The sample size was 20 µL. The mobile phase was pure methanol. The flow rate was 0.5 mL/min. The detector wavelength was 295 nm for sample concentrations below 0.3 g/L and 315 nm for sample concentrations in the range of 0.3-3 g/L. Pulse Tests. The pulse tests were performed with the semipreparative Chiralpak AD column using the HPLC pumps and PDA detector. The total bed void fraction was obtained by a pulse of 1,3,5-tri-tert-butylbenzene (tracer)dissolved in pure methanol. The tracer pulse size was 5 µL, and the tracer was monitored at 220 nm. The tracer was also used to measure the
Ind. Eng. Chem. Res., Vol. 42, No. 17, 2003 4059 Table 1. Fresh Solvent Costs39 solvent
cost ($/L)
methanol ethanol IPA heptane hexane ethyl acetate tetrahydrofuran toluene acetonitrile
0.18 0.74 0.59 0.32 0.30 1.19 3.00 0.30 1.13
extracolumn dead volume of the HPLC system without the column. The concentration of the racemic FTC-ester used in the pulse tests was 50 g/L. The pulse size ranged from 5 µL to 5 mL, and the mobile phase flow rate ranged from 0.5 to 1.5 mL/min. SMB Experiments. Before each SMB experiment, all eight columns were checked with FTC-ester pulses to ensure that the columns had consistent capacities and efficiencies. The concentration of the pulse was 50 g/L, the pulse size was 20 µL, and the mobile phase flow rate was 1 mL/min. The SMB experiment was started by turning on the pumps and triggering the timer of the valve controller (LabView) simultaneously. The extract and raffinate were collected over the period of the step time (between two consecutive valve switchings). The samples were taken from the collected extract and raffinate for HPLC assay. After the system reached cyclic steady state, the pumps were shut down, and column profile samples were taken from the bottom of each column. After each SMB experiment, each column was washed with pure methanol at a flow rate of 1 mL/min for 1 h. 4. Results and Discussion 4.1. Solvent-Sorbent Screening. The first step of the experimental work was a crude screening of the typical solvents used with chiral stationary phases. The solubilities of FTC and FTC-ester were estimated for several common HPLC solvents. The solubility of the FTC-ester was found to be roughly 3 times higher than that of the FTC for a given solvent composition. Therefore, we selected the FTC-ester for further study because of the higher solubility and the ease of integration into the processing steps. FTC and FTC-ester were more soluble in methanol than in the other solvents tested in this study. The solubility of FTC-ester in methanol was 15 wt % (FTCester/methanol). FTC and FTC-ester did not dissolve in pure heptane, hexane, or toluene. Compared with the other solvents, methanol is less costly and more environmentally benign. Furthermore, the solvent cost is a major fraction of the purification cost. The solvent prices listed in Table 1 indicate that methanol is the most economical solvent.39 Methanol was, therefore, selected as a good solvent for this study. Chiralpak AD, a chiral stationary phase (CSP), has been widely used because it is selective to many enantiomers.10,11,40 According to the manufacturer, methanol is compatible with this CSP. Therefore, Chiralpak AD and methanol were tested for the separation of FTCester in this study. Another mobile phase/CSP pair was also investigated for the FTC-ester separation. The mobile phase in this case was a mixture of toluene and THF, and the stationary phase was Kromasil A ¨ lgen, a product of Eka Chemicals (Bohus, Sweden). As the fraction of THF in
Table 2. Comparison of Two Mobile Phase/CSP Pairs Based on Preliminary SMB Design Kromasil A ¨ lgen and toluene/THF
Chiralpak AD and methanol
selectivity
2.4
solvent CSP equipment total
costa ($/kg of product) 133 13 43 189
2.6 29 11 43 83
a Assumptions in cost analysis: (1) Production rate ) 10,000 kg/year; (2) down time ) 20%; (3) CSP ) $10,000/kg (depreciated over 3 years); (4) solvent prices ) methanol $0.28/L, toluene/THF (55:45 v/v) $1.52/L; (5) solvent recycle ratio ) 80%; (6) SMB equipment ) $3,000,000 (depreciated over 7 years); (7) costs of labor, utilities, and waste disposal are excluded.
Table 3. Summary of Chiralpak AD Column Properties and Isotherm Parameters Column Properties length (cm)
column diameter (cm)
resin particle diameter (µm)
b
p
10
1
27
0.24
0.60
Isotherm Parameters
cis-(+)-FTCester cis-(-)-FTCester concentration range (g/L)
ab b ab b cis-(+) cis-(-)
isotherm A (racemic pulses)
isotherm B (enantiomer frontals)
isotherm Ca (SMB)
0.932 0.0055 2.43 0.085
