DE!%LINATION

EJSEVIER Desalination 148 (2002) 159-l 64 www.elsevier.com/locate/desal

pH and salt effects on chiral separations using affinity ultrafiltration

Jonathan Romero”, Andrew L. Zydneyb*

“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. +I (814) 863-7113, Fax +I (814) 8657846; email: zydney@engrpsu.edu

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 enautiomers [1,2]. Several recent studies [3-61 have demonstrated

*Corresponding author.

that affinity ultrafiltration, in which a large stereo- selective 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.

OOl l-9164/02/$- See front matter 0 2002 Elsevier Science B.V. All rights reserved PII: SO0 I l-9 164(02)0067 1 -Y

160 J. Romero, A.L. Zydney/Desalination I48 (2002) 159-164

requires multiple interactions between the macro- ligand and substrate in a very specific geometric orientation. The extent of binding is thus a strong function of solution pH and ionic strength, both of which can alter the magnitude of the underlying forces and change the geometric conformation of the macroligand and/or substrate.

McMenamy et al. [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). Maximum binding was observed around pH 9. Additional studies performed with tryptophan analogues showed a strong dependence on NaCl concentration, de- monstrating the importance of electrostatic inter- actions on the binding [9-l 11. 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 [ 12,131. Higuchi et al. [ 14,151 demonstrated the feasibility of using affinity ultrafiltration for the separation of D- and L-tryptophan based on the stereospecific binding of the L-stereoisomer by bovine serum albumin. Data were only obtained at pH 7, which was well removed from the maximum binding conditions reported by McMenamy et al. [ 81. Poncet et al. [3] and Gamier et al. [4] studied the binding of L- and D-tryptophan over a range of pH, with these binding curves used to evaluate the purity and recovery for an affinity ultrafiltration process. Unfortunately, no details were provided on how these calculations were performed, and the analysis appears to ignore the inherent changes in filtrate and retentate concentrations that occur during the membrane process. The maximum purity of D- tryptophan in the filtrate solution was achieved at pH 9, although these conditions gave a much lower D-tryptophan recovery than that obtained at pH <9 or at pH 11. No calculations were provided for the yield or recovery of the L-stereo- isomer.

These studies have clearly demonstrated the importance of solution conditions, and in particular solution pH, on stereospecific binding in affinity ultrafiltration processes for chiral separations.

However, 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. In this study, binding data were obtained for L- and D-tryptophan using bovine serum albumin as the stereoselective macroligand. These results were then used to explore the performance of an affinity diafiltration process for tryptophan separation, using purification - yield diagrams to determine the optimal process conditions.

2. Materials and methods

Binding data were obtained in a 25mm dia- meter stirred ultrafiltration cell (Model 8010, Amicon Corporation, Beverly, MA) using a 30,000 molecular weight cut-off polyethersulfone membrane that was fully retentive to the BSA. Filtrate samples were analyzed using capillary electrophoresis with a-cyclodextrine as the chiral resolving agent. 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. Addi- tional details on the experimental methods are pro- vided by Romero and Zydney [6].

3. Results and analysis

3. I. Binding interactions

Representative electropherograms of the feed (obtained prior to addition of BSA) and filtrate solutions are shown in Fig. 1 for a 0.4 mM racemic mixture of the L- and D-tryptophan in 1 mM NaCl with 0.6 mM added BSA at pH 5.3 and 8.5. The filtrate samples were collected shortly after pressurization of the stirred cell; thus, the retentate concentrations of the D- and L-tryptophan in the stirred cell were equal. The tryptophan concentra- tions were determined from the area under the electropherograms, giving a feed with Cr,/C, = 0.99 f 0.06 (as expected for the racemic mixture). Filtrate samples obtained in the absence of BSA were identical to the feed solution, indicating that

J. Romero, A.L. Zydney / Desalination 148 (2002) 1.59-164 161

1.4 I I

p 08 / fi,trate /yTrp D-Trp n 1

a 0.6 g

g

1000 1020 1040 1060 1080 1100

Electromigration Time (seconds)

Fig. 1. Electropherograms for initial feed and filtrate solutions (pH 5.3 and 8.5) at 22°C and 1 mM NaCI.

the free tryptophan is able to pass unhindered through the membrane. The filtrate samples at pH 5.3 and 8.5 showed significantly higher concen- trations of D-tryptophan due to the stereoselective binding of the L-tryptophan by BSA. The data at pH 5.3 gave C,,IC, = 0.99 with C’IC, = 0.77. The corresponding values at pH 8.5 were C,JCrD = 0.60 with C,lC, = 0.07. Thus, the extent of both L- and D-tryptophan binding was significantly greater at the higher pH, consistent with previous results reported by McMenamy et al. [8] and Poncet et al. [3].

The data in Fig. 1 were used to evaluate the fractional tryptophan binding:

(1)

where C,, and C, are the filtrate and total concentrations of either the L- or D-enantiomer. In each case, the total concentration was evaluated directly from the known mass of tryptophan added to the solution mixture. The results are shown in Fig. 2 using data for 0.4 mM racemic solutions with 0.6 mM BSA. The extent of tryptophan

lr’ I I I I t I I 1

1 *- c 0.8 - 0 ‘S

:

k 0.6 - c 2 z! x 0.4 - P t-

? ; 0.2 - a

mMNaCl a- m

w

A A A

A

A

A A A

A A

A D-Trp A

4 5 6 7 8 9 10 11 12

PH

Fig. 2. Binding fractions U;) vs. solution pH for D- and L- tryptophan in 1 mM NaCl (CT, = CT, = 0.2 mM, CmA = 0.6 mM).

binding increases with pH for pH ~8, with a maximum in the extent of binding for both the L- and D-enantiomers between pH 8 and 10. Thus, an affinity ultrafiltration process run in this pH range will yield a filtrate solution that is relatively dilute in both enantiomers, but with much less L- tryptophan passing through the membrane due to the stereospecific binding by BSA.

3.3. Purification factor - yield analysis

As discussed by Romero and Zydney [6,7], 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. This allows one to continually wash the less bound D-tryptophan through the membrane while maintaining a constant concen- tration of the large affinity ligand in the feed reservoir [6]. Under conditions where the binding fractions remain essentially constant as the less bound enantiomer is removed, the concentration of each enantiomer can be evaluated from a simple mass balance as described by Romero and Zydney [6,7]. The results are conveniently expressed in

162 J. Romero, A.L. Zydney / Desalination 148 (2002) 159-164

terms of the yield and purification factor for L- tryptophan in the retentate solution [ 161:

yL = “TL /

vc = exP[- ND Cl- fL >I TLo (2)

p = ( vc%TL~)

L ( vc%TLk.)

= exd% (fL - f. >I (3)

where N, is the number of diavolumes, which is equal to the ratio of the total collected filtrate volume (V’,) to the constant feed volume (V). The corresponding equations for the purification of D-tryptophan in the filtrate solution at constant6 are [16]:

Y, =VIC,=l-exp[-N,(l-fD)] J%kl

(4)

(5)

1 - ed- N, Cl- A, >I

= l-exp[-N,(l-f,)]

where V, is the total collected filtrate volume and c,; is the average concentration of D- or L-

tryptophan in the pooled filtrate obtained during the dia-filtration. The variation of the purification factor and yield throughout the diafiltration are described in more detail by Romero and Zydney

1671. Figs. 3 and 4 show plots of the yield and purifi-

cation factor for D-tryptophan and L-tryptophan, respectively, as a function of the number of dia- volumes at several pH. The solid curves represent the model calculations developed using Eqs. (2) and (3) for L-tryptophan and Eqs. (4) and (5) for

l pH 7.71 - Analytical soln. II

0 0.5 1 1.5 2 2.5

Number of Diavolumes, NI,

3

Fig. 3. Yield and purification factor for D-tryptophan during a constant volume aftinity diafdtration process as a function of the number of diavolumes. Solid curves are model calculations using CrD,,=CTLo= 0.2 mM and C,, = 0.6 mM at pH ranging from 4.8 to 11.4. Filled symbols are experimental data obtained during an actual diafiltration process at pH 7.71.

D-tryptophan based on the experimentally deter- mined values of the binding fractions at each pH from Fig. 2. The filled symbols represent data obtained by performing an actual single-stage dia-

J. Romero, A.L. Zydney / Desalination 148 (2002) 159-164 163

80

i

20

0 0.5 1 1.5 2 2.5 3

Number of Diavolumes, N D

Fig. 4. Yield and purification factor for L-tryptophan during a constant volume affinity diafiltration process as a function of the number of diavolumes. Solid curves are model calculations using CT,,,,= CT*,= 0.2 mM and C,,, = 0.6 mM at pH ranging from 4.8 to 11.4. Filled symbols are experimental data obtained during an actual diafiltration process at pH 7.71.

filtration at pH 7.71 and 1 mM NaCl (C,, =

0.6 mM, C,,= 0.2 mM). The scatter in the data arises from the inherent errors in the capillary electro- phoresis used to evaluate the tryptophan concen-

trations. The data are in fairly good agreement with the model predictions at this pH, demon- strating that the assumption of constant f, is appropriate in this system. The effects of a variablex on the affinity ultrafiltration have been discussed by Romero and Zydney [6,7,17]. The initial purification factor for D-tryptophan (Fig. 3) is highest at pH 9.2, but the yield of D-tryptophan remains quite low for this diafiltration due to the significant binding of this enantiomer under these experimental conditions (& = 0.39). Much higher yields are obtained at very high and low pH (e.g. pH 11.4 and pH 4.9), but the purification factors under these conditions remain less than 1.5 and decrease even further after larger numbers of dia- volumes. The corresponding results for the purifi- cation factor and yield for L-tryptophan in the retentate are shown in Fig. 4. The solid symbols again represent results from a diafiltration performed at pH 7.71, with the retentate concentrations evaluated from the measured filtrate concentrations using an overall mass balance. In this case, the process begins with a purification factor of P,=l since all of the tryptophan is initially present in the retentate reservoir. Both the yield and purifica- tion factor for L-tryptophan are highest at pH 9.2 due to the large values offL [see Eq. (2)] andf,-

fD [see Eq. (311.

4. Conclusions

Although previous studies have demonstrated that solution pH can have a large effect on stereospecific binding interactions, 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 purifi- cation of single enantiomers. 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. For the BSA - tryptophan system, the very strong binding of L-tryptophan around pH 9.2 leads to the highest purification

164 J. Ronzero, A.L. Zydney /Desalination 148 (2002) 159-164

factors and yield for this enantiomer, but these conditions actually lead to a low D-tryptophan yield in the filtrate due to the increase in the non- stereospecific binding for this enantiomer. In order to understand these effects more in detail, one can construct purification factor-yield plots [6,7,16] that can be used for process optimization. Note that it would also be possible to stage the mem- brane systems to achieve even higher purification factors and yields [ 171, although this would involve significantly greater capital investment for the membrane system. These phenomena will be considered in some detail in a future publication.

Symbols

cil - Filtrate concentration of D- or L-trypto-

c,,

phan M - Average concentration in the filtrate

collected over the diafiltration, M

c, - Total concentration (free+bound) of D- or L-tryptophan, M

c Tl,l - Initial total concentration of D- or L- tryptophan in feed solution, M

x - Fraction of bound D- or L-tryptophan

NLJ - Number of diavolumes

PI - Purification factor of D- or L- trypto- phan

V - Volume of retentate reservoir, m3 V

ri

- Total volume of collected filtrate, rn’ - Yield of D- or L-tryptophan

References

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M. Cayen, Chirality, 3 (1991) 94-98. R. Sheldon, Chiraltechnology: Industrial Synthesis of Optically Active Compounds. Marcel Dekker, New York, 1993. S. Ponce& J. Randon and J. Rocca, Sep. Sci. Tech., 32 (1997) 2029. F. Gamier, J. Randon and J-L. Rocca, Sep. Purif. Technol., 16 (1999) 243. J. Randon, F. Gamier, J. Rocca and B. Mdisterrena, J. Membr. Sci., 175 (2000) 111. J. Romero andA.L. Zydney, Sep. Sci. Tech., 36 (2001) 1575. J. Romero and A.L. Zydney, Biotechnol. Bioeng., 77 (2001) 256. R. McMenamy and J. Oncley, J. Biol. Chem., 233 (1958) 1436. R. McMenamy,Arch. Biochem. Biophys., 103 (1963) 409. R. McMenamy and R. Seder, J. Biol. Chem., 238 (1963) 3241. R. McMenamy, J. Biol. Chem., 239 (1964) 2835. W. Muller, K. Fehske and S. Schlafer, in M. Reidenberg and S. Erill (Eds.), Drug-Protein Binding. Praeger Publishers, New York, 1986. X. He and D. Carter, Nature, 358 (1992) 209. A. Higuchi, Y. Ishida andT. Nakagawa, Desalination, 90 (1983) 127. A. Higuchi, M. Hara, T. Horiuchi and T. Nakagawa, J. Membr. Sci., 93 (1994) 157. R. van Reis and S. Saksena, J. Membr. Sci., 129 (1997) 19. J. Romero and A.L. Zydney, J. Membr. Sci., (submitted).