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Desalination 148 (2002) 159–164

pH and salt effects on chiral separations using affinity ultrafiltration
Jonathan Romeroa, Andrew L. Zydneyb*
b a Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA Tel. +1 (814) 863-7113, Fax +1 (814) 865-7846; email:

Received 3 February 2002; accepted 15 March 2002

Abstract Affinity ultrafiltration can provide stereoselective separations using a large macroligand to selectively bind and retain one of the enantiomers. The detailed binding interactions can be strongly influenced by the solution pH and salt concentration, although there is currently little understanding of the impact of these phenomena on the overall performance of the affinity ultrafiltration process. Experimental studies were performed using a model system with bovine serum albumin as the stereoselective macroligand for the separation of D- and L-tryptophan. Binding data were obtained over a range of pH and ionic strength, with the results used to calculate the yield and purification factor during a diafiltration process using a recently developed model. L-tryptophan binding was greatest between pH 7 and 10, leading to a significant increase in purification within this pH range. Overall system performance, including the trade-offs between purification factor and yield, was examined by constructing purification factor — yield diagrams for the affinity ultrafiltration process. Keywords: Affinity ultrafiltration; Diafiltration; Ultrafiltration; Chiral separations; Stereoselectivity

1. Introduction There is considerable commercial interest in the production of single enantiomer products for the pharmaceutical, agricultural, and food industries due to the large differences in biological properties that often exists for different enantiomers [1,2]. Several recent studies [3–6] have demonstrated
*Corresponding author.

that affinity ultrafiltration, in which a large stereoselective ligand is used to bind and selectively retain one of the enantiomers, can be an attractive approach for large-scale commercial purification of enantiomers. The performance of any affinity ultrafiltration process is strongly affected by the detailed binding interactions between the macroligand and the product/impurities [6,7]. Stereospecific binding

Presented at the International Congress on Membranes and Membrane Processes (ICOM), Toulouse, France, July 7–12, 2002. 0011-9164/02/$– See front matter © 2002 Elsevier Science B.V. All rights reserved

The filtrate samples were collected shortly after pressurization of the stirred cell. The extent of binding is thus a strong function of solution pH and ionic strength.6 mM added BSA at pH 5. No calculations were provided for the yield or recovery of the L-stereoisomer. demonstrating the importance of electrostatic interactions on the binding [9–11]. [4] studied the binding of L. The maximum purity of Dtryptophan in the filtrate solution was achieved at pH 9. Additional studies performed with tryptophan analogues showed a strong dependence on NaCl concentration. Higuchi et al. there is currently no clear understanding of how to determine the optimal conditions for conducting the affinity ultrafiltration process based on these changes in binding interactions.and L-tryptophan in the stirred cell were equal. MA) using a 30. and in particular solution pH. no details were provided on how these calculations were performed. with these binding curves used to evaluate the purity and recovery for an affinity ultrafiltration process. thus.000 molecular weight cut-off polyethersulfone membrane that was fully retentive to the BSA. [14.06 (as expected for the racemic mixture). Maximum binding was observed around pH 9.4 mM racemic mixture of the L. These studies have clearly demonstrated the importance of solution conditions. Romero. Materials and methods Binding data were obtained in a 25-mm diameter stirred ultrafiltration cell (Model 8010. Additional details on the experimental methods are provided by Romero and Zydney [6]. and the analysis appears to ignore the inherent changes in filtrate and retentate concentrations that occur during the membrane process. Amicon Corporation. A. Filtrate samples were analyzed using capillary electrophoresis with α-cyclodextrine as the chiral resolving agent. More recent work has localized the L-tryptophan binding site to the IIIA subdomain in what is usually referred to as the indole-benzodiazepine site [12. giving a feed with CTL/CTD = 0. [3] and Garnier et al. Binding interactions Representative electropherograms of the feed (obtained prior to addition of BSA) and filtrate solutions are shown in Fig. These results were then used to explore the performance of an affinity diafiltration process for tryptophan separation.5.and D-tryptophan over a range of pH. [8]. Poncet et al.1. Results and analysis 3. Actual tryptophan separations were performed using a diafiltration process in which fresh buffer was added to the stirred cell to maintain a constant BSA concentration. Data were only obtained at pH 7. 1 for a 0. [8] performed a series of detailed studies of the effects of solution pH on the binding of the amino acid L-tryptophan to human serum albumin (HSA). which was well removed from the maximum binding conditions reported by McMenamy et al. on stereospecific binding in affinity ultrafiltration processes for chiral separations.160 J.L. Beverly. The tryptophan concentrations were determined from the area under the electropherograms. Zydney / Desalination 148 (2002) 159–164 requires multiple interactions between the macroligand and substrate in a very specific geometric orientation.99 ± 0. Filtrate samples obtained in the absence of BSA were identical to the feed solution. In this study. the retentate concentrations of the D. 2. binding data were obtained for Land D-tryptophan using bovine serum albumin as the stereoselective macroligand. both of which can alter the magnitude of the underlying forces and change the geometric conformation of the macroligand and/or substrate.3 and 8.15] demonstrated the feasibility of using affinity ultrafiltration for the separation of D. Unfortunately. 3.and L-tryptophan based on the stereospecific binding of the L-stereoisomer by bovine serum albumin. However. although these conditions gave a much lower D-tryptophan recovery than that obtained at pH <9 or at pH 11. using purification — yield diagrams to determine the optimal process conditions.and D-tryptophan in 1 mM NaCl with 0. McMenamy et al. indicating that .13].

6 mM).5 were CfD/CTD = 0. the concentration of each enantiomer can be evaluated from a simple mass balance as described by Romero and Zydney [6. [3].7].3 L-Trp D-Trp 0. Purification factor — yield analysis As discussed by Romero and Zydney [6.2 D-Trp feed 0 1000 1020 1040 1060 1080 1100 0 4 5 6 7 8 pH 9 10 11 12 Electromigration Time (seconds) Fig. The results are shown in Fig.4 0. The extent of tryptophan .5 i D-Trp 1 mM NaCl 0. 1. The corresponding values at pH 8. 2 using data for 0. the affinity ultrafiltration is best conducted using a diafiltration mode in which fresh buffer is added to the feed reservoir at the same rate at which the filtrate is removed. 1 were used to evaluate the fractional tryptophan binding: fi = 1 − C fi CTi (1) binding increases with pH for pH <8. [8] and Poncet et al.8 0. the free tryptophan is able to pass unhindered through the membrane.4 161 1 Absorbance (arbitrary units) L-Trp D-Trp L-Trp Bound Tryptophan Fractions.6 filtrate pH 5. The data at pH 5. an affinity ultrafiltration process run in this pH range will yield a filtrate solution that is relatively dilute in both enantiomers.2 1 0. Thus. f 1.77.5 showed significantly higher concentrations of D-tryptophan due to the stereoselective binding of the L-tryptophan by BSA.4 mM racemic solutions with 0.6 mM BSA. Fig. Under conditions where the binding fractions remain essentially constant as the less bound enantiomer is removed.60 with CfL/CTL = 0. Binding fractions (fi) vs.and D-tryptophan binding was significantly greater at the higher pH. solution pH for D. but with much less Ltryptophan passing through the membrane due to the stereospecific binding by BSA. the extent of both L.8 L-Trp 0.2 filtrate pH8. In each case.2 mM. with a maximum in the extent of binding for both the Land D-enantiomers between pH 8 and 10. Zydney / Desalination 148 (2002) 159–164 1. consistent with previous results reported by McMenamy et al.99 with CfL/CTL = 0.7]. The data in Fig.3 and 8.6 0.J.4 0.or D-enantiomer. the total concentration was evaluated directly from the known mass of tryptophan added to the solution mixture.5) at 22°C and 1 mM NaCl.3 and 8.3. Thus. Romero. 2. 3. This allows one to continually wash the less bound D-tryptophan through the membrane while maintaining a constant concentration of the large affinity ligand in the feed reservoir [6]. Electropherograms for initial feed and filtrate solutions (pH 5.L.07. The filtrate samples at pH 5.3 gave CfD/CTD = 0. A.and Ltryptophan in 1 mM NaCl (CTD = CTL = 0. CBSA = 0. The results are conveniently expressed in where C fi and C Ti are the filtrate and total concentrations of either the L.

29 11.2 mM and CBSA = 0. Yield and purification factor for D-tryptophan during a constant volume affinity diafiltration process as a function of the number of diavolumes.71 7.45 11 V f C fD PD = V f C fL 60 VCTDo VCTLo 40 (5) 1 − exp[− N D (1 − f D )] = 1 − exp[− N D (1 − f L )] 20 0 0 0.71. Zydney / Desalination 148 (2002) 159–164 9 terms of the yield and purification factor for Ltryptophan in the retentate solution [16]: YL = VCTL VCTLo = exp[− N D (1 − f L )] (2) D-tryptophan Purification factor.42 5. The filled symbols represent data obtained by performing an actual single-stage dia- .71 Analytical soln. P pH 7.71 9. N Fig.or Ltryptophan in the pooled filtrate obtained during the dia-filtration. The corresponding equations for the purification of D-tryptophan in the filtrate solution at constant fi are [16]: YD = V f C fD VCTDo = 1 − exp[− N D (1 − f D )] 1 100 A (4) D-tryptophan Yield. Y D 80 pH 11. 2. The variation of the purification factor and yield throughout the diafiltration are described in more detail by Romero and Zydney [6.6 mM at pH ranging from 4.98 pH 7. Figs. which is equal to the ratio of the total collected filtrate volume (Vf) to the constant feed volume (V). (2) and (3) for L-tryptophan and Eqs. Solid curves are model calculations using CTDo =CTLo = 0. respectively. 3 and 4 show plots of the yield and purification factor for D-tryptophan and L-tryptophan. Romero. as a function of the number of diavolumes at several pH.5 D 3 where Vf is the total collected filtrate volume and C fi is the average concentration of D.4. (4) and (5) for Number of Diavolumes.42 11 5.21 10. 7 VCTL PL = VCTD D VCTLo VCTDo = exp[N D ( f L − f D )] 5 pH 9. 3.21 10. The solid curves represent the model calculations developed using Eqs.98 where ND is the number of diavolumes.8 to 11.5 2 2.L. D-tryptophan based on the experimentally determined values of the binding fractions at each pH from Fig. A. Filled symbols are experimental data obtained during an actual diafiltration process at pH 7.162 J.45 (3) 3 7.5 1 1.7].

Solid curves are model calculations using CTDo = CTLo = 0.98 11.45 7. The scatter in the data arises from the inherent errors in the capillary electrophoresis used to evaluate the tryptophan concen- Although previous studies have demonstrated that solution pH can have a large effect on stereospecific binding interactions. Filled symbols are experimental data obtained during an actual diafiltration process at pH 7. but the purification factors under these conditions remain less than 1.L.4 and pH 4. Y L pH 9.J.g. the very strong binding of L-tryptophan around pH 9.4.6 mM at pH ranging from 4.9). A. the process begins with a purification factor of PL=1 since all of the tryptophan is initially present in the retentate reservoir. P 163 pH 7. The corresponding results for the purification factor and yield for L-tryptophan in the retentate are shown in Fig. 3) is highest at pH 9.2 due to the large values of fL [see Eq.21 10. pH 9.71 11 5.45 7. Conclusions L 0 0 0.71 and 1 mM NaCl (CBSA = 0. demonstrating that the assumption of constant fi is appropriate in this system.71 11 60 40 5.71.71 Analytical soln.17]. Zydney / Desalination 148 (2002) 159–164 10 L-tryptophan Purification Factor.5 Number of Diavolumes.5 2 2.2 mM and CBSA = 0. N D 3 Fig. Yield and purification factor for L-tryptophan during a constant volume affinity diafiltration process as a function of the number of diavolumes. 4. The data and analysis presented in this manuscript provide an initial framework that can be used to analyze affinity diafiltration using independent binding data obtained for the two enantiomers with a specific affinity macroligand. 4. (3)]. pH 11.2.6 mM. Both the yield and purification factor for L-tryptophan are highest at pH 9. 4.2 mM). (2)] and fL – fD [see Eq.42 trations. Romero. but the yield of D-tryptophan remains quite low for this diafiltration due to the significant binding of this enantiomer under these experimental conditions (fD = 0.98 20 11. CTio= 0. with the retentate concentrations evaluated from the measured filtrate concentrations using an overall mass balance.8 to 11.21 10.71.42 1 100 80 L-tryptophan Yield. For the BSA – tryptophan system.7. The data are in fairly good agreement with the model predictions at this pH. The solid symbols again represent results from a diafiltration performed at pH 7.39). In this case.2 leads to the highest purification .5 and decrease even further after larger numbers of diavolumes. The effects of a variable fi on the affinity ultrafiltration have been discussed by Romero and Zydney [6.5 1 1. Much higher yields are obtained at very high and low pH (e. filtration at pH 7. The initial purification factor for D-tryptophan (Fig. there has been little understanding of how to properly translate these results into an analysis of the performance of an affinity ultrafiltration process for the purification of single enantiomers.

Chem. J. although this would involve significantly greater capital investment for the membrane system. 93 (1994) 157. Membr. McMenamy.L.. CTi CTio fi ND Pi V Vf Yi — Filtrate concentration of D. 3 (1991) 94–98. Zydney / Desalination 148 (2002) 159–164 factors and yield for this enantiomer. 239 (1964) 2835. J. Rocca. Sep. [7] J. [6] J. Carter.. Biophys. Sheldon. [12] W. Fehske and S.or Ltryptophan in feed solution. Nakagawa. Cayen.. In order to understand these effects more in detail. M — Total concentration (free+bound) of Dor L-tryptophan. 358 (1992) 209. Purif. Biol. [11] R.or L. Horiuchi and T. McMenamy and R. M — Initial total concentration of D.7. Nature. M — Average concentration in the filtrate collected over the diafiltration. J. Nakagawa. Sci. J. Y. 1986. [9] R. Chem. [10] R. Sep. [16] R. J. Reidenberg and S. 90 (1983) 127. Romero. J. Sci.or L-tryptophan . 129 (1997) 19. Biochem. 103 (1963) 409. Schlafer. Sci. Randon.L. Biol. Desalination. [5] J. Poncet. Zydney.. [13] X. Muller.). [2] R. 36 (2001) 1575. van Reis and S. Higuchi... 1993. one can construct purification factor — yield plots [6.L. Rocca and B.164 J. Tech. Chiraltechnology: Industrial Synthesis of Optically Active Compounds. McMenamy. Romero and A. Sci.16] that can be used for process optimization.or L-tryptophan. [4] F. Tech. [15] A. Chirality. New York. 77 (2001) 256. Praeger Publishers. Saksena.L. McMenamy and J.. Garnier. Sep. in M. Symbols Cfi C fi References [1] M. Oncley. J. [8] R.tryptophan — Volume of retentate reservoir. Biotechnol. Romero and A. Seder. 32 (1997) 2029. K. Biol. Marcel Dekker. Membr.. Sci. [14] A. New York. J. F. Hara. m3 — Total volume of collected filtrate. M. 175 (2000) 111. Garnier. Higuchi. These phenomena will be considered in some detail in a future publication. Zydney. J. Arch.. but these conditions actually lead to a low D-tryptophan yield in the filtrate due to the increase in the nonstereospecific binding for this enantiomer. Membr. [17] J. Bioeng. Sci. Zydney. [3] S.. Technol.or L-tryptophan — Number of diavolumes — Purification factor of D. Note that it would also be possible to stage the membrane systems to achieve even higher purification factors and yields [17]. He and D. Maïsterrena. Randon and J-L. 233 (1958) 1436. Chem. 16 (1999) 243. Erill (Eds. 238 (1963) 3241.. Romero and A. m3 — Yield of D. T. (submitted). M — Fraction of bound D.. J. Ishida and T. A. Membr. Rocca. Randon and J. Drug-Protein Binding.