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... Regeneration and Reusability of HSA as a Stereoselective Ligand for Chiral Separation in Affinity UF 3.1 Characterizations of hollow fiber membranes 28 3.2 Tryptophan separation of hollow fiber membranes... consisted of D-tryptophan inferring the preferential binding of L-tryptophan to BSA The separation factor of Dtryptophan over L-tryptophan as high as was attained, which demonstrates the feasibility of. .. 3.6 Comparison of yields of L-tryptophan (A) and D-tryptophan (B) between native (●) and recovered (○) HSA Figure 3.7 Comparison of tryptophan separation factor between native (●) and recovered
APPLICATION OF MEMBRANE TECHNOLOGIES FOR BIOMOLECULAR SEPARATION AND PURIFICATION BY AFFINITY BINDING TO SPECIFIC LIGANDS FELINIA (B.Eng., Bandung Institute of Technology) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements First of all, I would like to sincerely acknowledge my supervisor, Prof. Neal Chung Tai-Shung from the department of Chemical and Biomolecular Engineering, National University of Singapore for his guidance, suggestions, advices, and encouragements throughout my master study. Special thanks are given to Professor T. Alan Hatton from the Massachusetts Institute of Technology and Professor Raj Rajagopalan from the National University of Singapore for their invaluable suggestions. I also want to thanks the Singapore-MIT Alliance and the National University of Singapore for providing the research funding. Finally, I want to express my appreciation to my mentor, Dr. Li Yi and all of my colleagues in the membrane research group for their kind assistances. i Table of Contents Acknowledgments...................................................................................................... i Summary .................................................................................................................... iv List of Tables ............................................................................................................. v List of Figures ............................................................................................................ vi List of Symbols .......................................................................................................... vii Chapter 1. Background Review and Objectives 1.1 Introduction ................................................................................................... 1 1.2 Methodologies of enantiomers separation .................................................... 2 1.3 Introduction to membrane for enantiomers separation ................................. 4 1.3.1 Enantioselective membranes .......................................................................... 5 1.3.2 Non-enantioselective membranes ................................................................. 6 1.4 Introduction to affinity UF system ................................................................ 7 1.4.1 Affinity UF system performance parameters ................................................ 7 1.4.1.1 Enantioselectivity .......................................................................................... 7 1.4.1.2 Permeability ................................................................................................... 9 1.4.2 Development of affinity UF system .............................................................. 9 1.4.3 Selection of stereoselective ligand ................................................................ 12 1.5 Formation of hollow fiber membranes by phase inversion .......................... 14 1.6 Research objectives ....................................................................................... 16 Chapter 2. Experimental 2.1 Materials ....................................................................................................... 17 ii 2.2 Dope preparation ........................................................................................... 18 2.3 Hollow fiber spinning ................................................................................... 19 2.4 Morphology study by FESEM ...................................................................... 20 2.5 Contact angle measurements ......................................................................... 20 2.6 Module fabrications ...................................................................................... 20 2.7 PWP and pore size distribution characterizations ......................................... 21 2.8 Tryptophan separation performance ............................................................. 22 2.9 HSA denaturation and renaturation study ..................................................... 23 2.10 HSA regeneration and reusability ................................................................. 26 2.11 CD measurements of HSA ............................................................................ 26 Chapter 3. Exploration of Regeneration and Reusability of HSA as a Stereoselective Ligand for Chiral Separation in Affinity UF 3.1 Characterizations of hollow fiber membranes .............................................. 28 3.2 Tryptophan separation of hollow fiber membranes ...................................... 31 3.3 Tryptophan separation of native and renatured HSA ................................... 33 3.4 Tryptophan separation of native and recovered HSA ................................... 37 Chapter 4. Conclusion ............................................................................................ 40 Bibliography ............................................................................................................ 41 Appendix .................................................................................................................. 47 iii Summary The reusability of human serum albumin (HSA) as a stereoselective ligand for D,L- tryptophan separation in the affinity ultrafiltration (UF) system has been demonstrated by readjusting the medium pH from an acidic condition to a basic condition in this study. The native and recovered HSA molecules exhibit a similar D,L-tryptophan separation factor of 5-7 under the same experimental conditions. In addition, a high recovery percentage of HSA of above 80% has also been obtained by controlling both the membrane pore size and membrane hydrophilicity. The combination of these two features (i.e. HSA reusability and high recovery) is very helpful for the large-scale industrial application of the affinity UF system in chiral separation. On the other hand, it has been found that the HSA binding capability to L-tryptophan could be affected by the solution ionic strength. A higher solution ionic strength may result in a decrease in amounts of L-tryptophan bound to HSA due to the changes in solution environment and HSA structure. iv List of Tables Table 1.1 Comparison of existing enantioseparation techniques Table 3.1 Spinning parameters for hollow fiber membranes Table 3.2 Comparison of PWP and parameters from neutral solute rejection experiments Table 3.3 Overall separation factor, overall L-tryptophan yield and percentage of recovered HSA for fiber-15cm Table 3.4 Summary of far-UV CD and near-UV CD data for native, acid-denatured and base-renatured HSA with different denaturation-renaturation times v List of Figures Figure 1.1 Different techniques for separation of the enantiomers Figure 1.2 Separation mechanisms of enantioselective and non-enantioselective membranes Figure 1.3 Separation mechanism of D,L-tryptophan racemic mixtures with BSA as a stereoselective ligand. (A) D,L-tryptophan in BSA solution, (B) Ltryptophan bound to BSA, (C) L-tryptophan ejected from HSA Figure 1.4 Schematic of hollow fiber spinning Figure 2.1 Chemical structure of PES polymer Figure 2.2 Chemical structure of PVP Figure 2.3 Schematic diagram of hollow fiber spinning line Figure 2.4 Schematic diagram of self-designed UF setup for PWP and pore size distribution characterizations Figure 2.5 Schematic of HSA denaturation and renaturation experiments Figure 2.6 Schematic of tryptophan separation experiments for HSA denaturation and renaturation study under diafiltration mode Figure 3.1 Comparison of FESEM images of PES hollow fiber membranes spun at various air gap distances Figure 3.2 Cumulative pore size distribution (A) and probability density function (B) curves for hollow fiber membranes Figure 3.3 Capillary electropherograms of permeate samples via hollow fiber membranes Figure 3.4 Comparison of tryptophan separation factor of hollow fiber membranes Figure 3.5 Far-UV CD spectra (A) and near-UV CD spectra (B) for native, one time acid-denatured and one time base-renatured HSA Figure 3.6 Comparison of yields of L-tryptophan (A) and D-tryptophan (B) between native (●) and recovered (○) HSA Figure 3.7 Comparison of tryptophan separation factor between native (●) and recovered (○) HSA vi List of Symbols A membrane area (m2) cf neutral solute concentration in feed (mol/m3) Cf,D D-enantiomer Cf,HSA HSA concentration in feed (mol/m3) Cf,L L-enantiomer CHSA HSA concentration (mol/m3) cp neutral solute concentration in permeate (mol/m3) Cp,D D-enantiomer concentration in permeate (mol/m3) Cp,L L-enantiomer concentration in permeate (mol/m3) Cr,HSA HSA concentration in retentate (mol/m3) ee enantiomeric excess (%) J normalized flux (L/m2.hr.kPa) Jv permeate flux (m/s) K non-stereospecific complexation constant (-) KL stereospecific complexation constant (-) l cell pathlength (cm) MRE mean residue ellipticity (deg.cm2/dmol) MRE222 MRE at wavelength of 222 nm (deg.cm2/dmol) MW molecular weight (Da) n number of amino acid residues (-) nf,D D-enantiomer in feed (mol) nf,L L-enantiomer in feed (mol) np,D D-enantiomer in permeate (mol) concentration in feed (mol/m3) concentration in feed (mol/m3) vii np,L L-enantiomer in permeate (mol) Pm membrane permeability (m) rp membrane pore radius (nm) rs solute Stokes radius (nm) RT solute rejection (-) t sampling time (hour) V permeate volume (L) YD D-tryptpohan yield (%) YL L-tryptpohan yield (%) YL,o overall L-tryptpohan yield (%) % helix α-helical content (%) % Rec,HSA HSA recovery (%) µ feed viscosity (kg/m.s) µp mean pore radius (nm) µs geometric mean solute radius at RT = 50% (nm) α separation factor (-) αo overall separation factor (-) ΔP trans-membrane pressure drop (kPa) σp standard deviation (-) 𝜃obs CD measurement (mdeg) viii Chapter 1. Background Review and Objectives 1.1 Introduction Chirality is a phenomenon used to describe any objects existing in two forms that lack of plan, center, and axis of symmetry and thus, creating a non-superimposable mirror image to one another. Chirality can be easily observed in the daily life from human body until small molecules possessing asymmetric carbon. The chemistry terms for those compounds are stereoisomers or enantiomers or enantiomorphs or chiral molecules [1]. The term racemic mixture is referring to the enantiomer ratio of 50/50. In 1930, it was discovered that different enantiomers possess different biological activities depending on their affinities towards enzyme or other receptor [2]. The enantiomer that strongly binds to the active sites of the receptor is called eutomer, while the enantiomer with a weaker association to the receptor is called distomer. This finding has significant impacts in enantiomers used as reagents in pharmaceutical as the distomer may not only reduce the activity of the eutomer but may cause toxic side effects. For instance, the thalidomide was previously prescribed as a sedative and antiemietic drug while it was found out later on that its R-enantiomer can cause teratogenic effects [3]. Reflecting from the thalidomide tragedy, the U.S. Food and Drug Administration (FDA) established a new policy in 1992 for the development of new stereoisomeric drugs. It required every pharmaceutical company to identify the stereoisomeric composition of a drug with a chiral center together with the toxicological and pharmacological properties of the individual enantiomers [4]. The drugs need to be 1 administered in a pure enantiomer form if the other enantiomer is proven to possess different toxicological and pharmacological properties or any side-effects. This policy has created new markets for redevelopment of pure enantiomeric drugs that have been previously approved in a racemic form. Increasing public awareness of the possible hazard of racemic drugs has boast up the worldwide sales of the stereochemically pure drugs from 27% (US $74.4 billion) in 1996 to around 39% (US $151.9 billion) in 2002 [5]. To the present, there are two ways to produce single enantiomer drugs: synthesis and separation methods. Even though through the synthesis method, a high purity drug can be achieved yet its commercial application is still limited by the low yield. On the other hand, a high yield can be achieved through the separation method yet its cost inefficiency remains the limiting factor [6]. Therefore, the development of cost and time efficient enantioseparation methods for commercial production of the stereochemically pure drugs is urgently needed. 1.2 Methodologies of enantiomers separation Depends on its applications, enantiomers separation can be classified into two main categories: analytical and preparative methods. Analytical method refers to any enantioseparation techniques aiming to analyze the stereoisomeric composition of chiral compounds. In analytical scale, the short testing time and high selectivity and sensitivity are compulsory. In this regard, the chromatography and electrophoresis techniques are able to suffice such requirements and commonly selected for enantiomers analysis purpose [7]. 2 Meanwhile, the most important factors for the preparative method are the high loading capacity and selectivity in order to improve the productivity. In industrial scale, the chromatographic and crystallization techniques are the most popular methods while membrane is considered as an emerging approach in the field of enantioseparation [7]. Various methodologies for separation of enantiomers are schematized in Fig. 1.1 while the merits and drawbacks of the existing enantioseparation techniques are summarized in Table 1.1. Analytical Analytical/Preparative Figure 1.1 Different techniques for separation of the enantiomers [7] 3 Table 1.1 Comparison of existing enantioseparation techniques [19] No. Methods 1. Crystallization Advantages Simplicity 1.1 Direct or preferential crystallization [8-9] Low cost Batch operation 1.2 Diastereomeric crystallization [10-11] Kinetic resolution Wide applicability Expensive 2.1 Chemical-mediated [1213] High stability Low efficiency 2.2 Enzyme-mediated [1415] Chromatography High efficiency Degrading enzyme activity Low capacity 3.1 Supercritical fluid chromatography [16] Low cost 3.2 Simulated moving bed chromatography [17-18] Membrane [19-36] Continuous operation 2. 3. 4. Disadvantages High efficiency Low cost, energy efficient, high capacity, continuous operation, easy scaleup Low number of transfer units per apparatus 1.3 Introduction to membrane for enantiomers separation Membrane processes have gained much attention recently in the field of enantiomeric separation owing to their cost and energy efficiency, suitability for continuous operation, feasibility for up-scaling, and ease of modification to accommodate different process configurations [7, 19]. Generally, membrane-based enantioseparation can be classified into two types: enantioselective and nonenantioselective membranes [19-20]. 4 1.3.1 Enantioselective membranes In the enantioselective membrane-based separation process, the membrane possesses chiral recognition sites which can selectively bind one of the two enantiomers. Depends on the membrane configuration, enantioselective membrane can be further classified into solid and liquid membranes. Solid enantioselective membranes can be directly prepared from phase inversion of the chiral polymers which by default posses chiral recognition properties, such as polyamino acids, polysaccharides, or polypeptides [21-22]. Other alternatives to fabricate enantioselective membranes are mixing the stereoselective ligands into non-chiral polymer solution before membrane casting [23] or immobilized the chiral selectors on the surface or membranes pores by grafting or molecular imprinting techniques [24-25]. Separation mechanism for the solid enantioselective membrane can be categorized into two: diffusion-selectivity and adsorption-selectivity. The binding strength between the chiral recognition sites and enantiomers determines the separation mechanism, for example a weaker association is normally classified as a diffusionselectivity as it assists to slow down mass transport one of the enantiomers. As for the liquid enantioselective membranes, enantioseparation is facilitated by mobile chiral selectors dissolved in the liquid membrane phase which can selectively transport one of the enantiomers [26-27]. Liquid enantioselective membranes are gaining much interest owing to its low resistance for mass transfer compared to its solid counterpart. However, improvements on membrane durability are required for liquid membrane to withstand long-term separation. 5 1.3.2 Non-enantioselective membranes Different from the enantioselective membranes, the separation mechanism for nonenantioselective membranes is normally based on the size difference. As the enantiomers are typically identical in size, this process must be combined with other chiral recognition techniques to accomplish enantioseparation, for example catalyzed kinetic resolution [28-29] or enantiomer binding with stereoselective ligands [30-36]. The kinetic resolution is often catalyzed by enzymes, such as lipase to hydrolyze the enantiomers followed by membrane filtration to filter out the inhibitory byproducts. The differences in the separation principle between enantioselective and nonenantioselective membranes are illustrated in Fig 1.2. Figure 1.2 Separation mechanisms of enantioselective and non-enantioselective membranes 6 The hybrid enantioseparation method combining non-enantioselective membrane with enantiomer-ligand complex is often referred as affinity UF. The working principle of the affinity UF is that after one enantiomer is bound selectively onto a large stereoselective ligand added to the bulk solution, the enantiomer-ligand complex is retained by a membrane, while the unbound enantiomer is transported across the membrane and collected on the permeate side. It is obvious that in the affinity UF system, the enantioselectivity is governed by selection of the stereoselective ligand. It is important to mention that non-enantioselective membranes have received great attentions owing to its convenience to combine the merits of different separation techniques and its potencies for large-scale enantioseparation [19]. Therefore, the non-enantioselective membrane particularly the affinity UF system will be discussed in detail in the next section. 1.4 Introduction to affinity UF system 1.4.1 Affinity UF system performance parameters Similar to other membrane separation process, the performance of affinity UF system can be defined by two parameters, namely selectivity and permeability. Both high selectivity and permeability are required to maximize system productivity. 1.4.1.1 Enantioselectivity Enantioselectivity is an important factor in any separation system as it determines the purity of the final product. As for the affinity UF and other non-enantioselective membrane systems, the enantioselectivity is mostly attributed to the differences in the association between the seteroselective ligand and the two enantiomers. Generally, the stereoselective ligand possesses a stronger binding with one enantiomer developing 7 enantioselectivity yet non-stereospecific interactions may also occur through hydrogen bonding, hydrophobic, Coulombic, and van der Waals types of interaction [30]. Both enantiomers compete to bind to the non-stereospecific sites thus supressing the overall enantioselectivity. Many terms are used in the publications to define the enantioselectivity. The examples of the commonly used terms in the publications are separation factor (α) and enantiomeric excess (ee). The separation factor is defined as the concentration ratio between two enantiomers in the permeate solution divided by that in the feed solution [26]. The separation factor is calculated as follows: C p,D α= C p,L C f ,D (1.1) C f ,L where Cp,D and Cp,L are the concentrations of D-enantiomer and L-enantiomer in the permeate side, respectively, while Cf,D and Cf,L are the concentrations of D-enantiomer and L-enantiomer in the feed side, respectively. The separation factor is the most commonly used term for selectivity calculation in the affinity UF system. Therefore, for ease of comparison purposes, the selectivity presented in this study was calculated in terms of the separation factor. The enantiomeric excess is defined as the ratio of concentration difference in the permeation between two enantiomers divided by the concentration summation of both enantiomers [31]. The following equation is used to calculate the enantiomeric excess. = ee C p,D − C p,L C p,D + C p,L ×100 (1.2) 8 1.4.1.2 Permeability Another important factor that regulates the process productivity is permeability (Pm). As commonly observed in other porous membrane system, permeability is strongly dependent on membrane properties, such as membrane thickness, porosity, tortuosity, and pore size. With the trans-membrane pressure being the main driving force, the permeability (Pm) is calculated as follows: Pm = Jv × µ ∆P (1.3) where Jv is the permeate flux (m/s), µ is the feed viscosity (kg/m.s), and ΔP is the trans-membrane pressure (kPa). The permeability is also reported in terms of permeation flux (J) that is defined as: J= V A × t × ∆P (1.4) where V is the volume of the permeate collected (L), A is the membrane area (m2), and t is the sampling time (hour). 1.4.2 Development of affinity UF system The affinity UF system was firstly proposed in 1993 by Higuchi and coworkers [32]. They utilized 0.6 mM bovine serum albumin (BSA) as a stereoselective ligand for separation of 0.049 mM D,L-tryptophan racemic mixtures. The solution pH was varied from 7.0 to 3.0 and the effects on the enantioselectivity were investigated. It was reported that at pH 7.0, the permeate solution was mainly consisted of D-tryptophan inferring the preferential binding of L-tryptophan to BSA. The separation factor of Dtryptophan over L-tryptophan as high as 7 was attained, which demonstrates the feasibility of an affinity UF system for chiral separation. Interestingly, when the 9 solution pH was changed to 3.0, L-tryptophan was existed in the permeate solution indicating detachment of the L-tryptophan from the BSA binding sites at low pH. However, some important parameters were not reported in the manuscript, such as permeability and yield. The enantioseparation mechanism of D,L-tryptophan racemic mixtures with incorporation of BSA as a stereoselective ligand is depicted in Fig. 1.3. Figure 1.3 Separation mechanism of D,L-tryptophan racemic mixtures with BSA as a stereoselective ligand. (A) D,L-tryptophan in BSA solution, (B) L-tryptophan bound to BSA, (C) L-tryptophan ejected from HSA [32] Randon’s group [33] further investigated the association between BSA and the D,L- tryptophan racemic mixture. They designed experiments to determine the stereospecific and non-stereospecific complexation constants between L-tryptophan and BSA at different solution pH. The relations employed are as follows: • Non-stereospecific complexation constant BSA + L ↔ BSA-L BSA + D ↔ BSA-D [ BSA-L] [ BSA ][ L] [ BSA-D] K= [ BSA ][ D] K= (1.5) (1.6) • Stereospecific complexation constant 10 [S-L] [S][ L] [ BSA ]0 = [ BSA ] + [ BSA-L] + [ BSA-D] [ D]0 = [ D] + [ BSA-D] [ L]0 = [ L] + [ BSA-L] + [S-L] S + L ↔ S-L KL = (1.7) (1.8) (1.9) (1.10) They reported that the stereospecific complexation constant reached a maximum point at the solution pH of 9.0 (KL = 110,000) whereas the non-stereospecific complexation constant reached a maximum point at pH of 10.0 (K = 3,500). The enantioseparation of D,L-tryptophan racemic mixtures was then performed with the BSA solution. The highest D-tryptophan purity and recovery of 91% and 89%, respectively was attained at the solution pH of 9.0. However, there was not any detail provided on the tested system to achieve the enantioseparation reported in the mansucript. Afterwards, Zydney’s group [34-36] developed the affinity UF system through optimization of the single-stage UF system operating conditions and employment of multistage designs. Diafiltration system in which the fresh buffer was topped up into the feed solution to balance the loss of the permeate solution was proposed to eliminate the problems associated with the concentrated BSA in the feed. The Ltryptophan yield was reported as a function of diavolumes which was obtained by dividing the volume of the collected permeates with the initial feed volume. By employing 0.6 mM BSA and 0.3 mM D,L-tryptophan racemic mixture dissolved in buffer solution with pH of 8.5, the highest separation factor of D-tryptophan over Ltryptophan of around 11.0 was achieved at the beginning of the experiment. The separation factor was dropped to around 6.0 when the diavolumes was equal to 3. The authors explained that the degradation in the separation factor was caused by dilution 11 of the D-tryptophan remaining in the feed. Moreover, they also have demonstrated that the separation factor can be further increased to around 20.0 by employing the twostage affinity UF system. 1.4.3 Selection of stereoselective ligand Generally, the criteria for selection of the stereoselective ligand in the affinity UF system are as follows: (1) a high intrinsic selectivity, (2) a much larger size compared to the enantiomers and hence the enantiomer-ligand complex can be easily retained by the UF membrane, and (3) feasibility to be regenerated and reuse. In most of the reported studies, tryptophan was commonly employed as the model enantiomers with various stereoselective ligands incorporated to achieved enantioseparation. The examples of the commonly used stereoselective ligands are cyclodextrins [37], deoxyribonucleic acid (DNA) [38], L-glutamic acid derivatives [39], and albumins [40]. Albumin is a globular protein consisting of 585 amino acid residues and serves as a major transport protein in the circulatory system [41-42]. Up to the present, the most widely used albumin in the affinity UF system is albumin obtained from bovine (BSA). However, the difference in D,L-tryptophan association constants of BSA and HSA has been reported [43]. The L-tryptophan and D-tryptophan association constants for BSA are 1.5×10-4 M-1 and 0.23 ×10-4 M-1, respectively, while the L-tryptophan and D-tryptophan association constants for HSA are 1.1× 10-4 M-1 and 0.13 ×10-4 M-1, respectively. Thus, the intrinsic selectivity can be approximated from the ratio of association constants between the stereoselective ligand and two enantiomers. In this 12 regard, HSA possesses a higher intrinsic selectivity of L-tryptophan over D-tryptophan compared to BSA. HSA has been known to have two principal binding sites; namely, the warfarinazapropazone site (site I) and the indol-benzodiazepine site (site II) [44-45]. The characterization of these two binding sites by the displacement of fluorescent probe molecules from HSA by drugs has been done and it was suggested that electrostatic and dipolar forces contribute to the HSA specificity and binding affinity [45]. Later on, the binding chemistry of the site II of HSA in which the L-tryptophan is selectively bound was investigated through a series of studies by Peyrin et al. [46-49]. The binding mode was described in which the nonpolar residues inside the binding cavity of HSA were occupied by the hydrophobic groups of the bound molecule, while the cationic and polar residues of the cavity rim interacted with the carboxylate and sulfonalimido groups of the bound molecule. Therefore, the HSA selectivity towards L-tryptophan is determined by the arrangement of the hydrophobic functionalities together with the carboxylate and sulfonalimido groups of the Ltryptophan molecule. Unless, a molecule possesses those groups in an arrangement fitted the binding cavity of HSA, it will not be attached to the stereospecific binding site of HSA. Moreover, the HSA selectivity towards L-tryptophan is also dependent on the HSA conformation as the dimension and structure of the binding cavity may change as the HSA conformation is disrupted leading to a loss of the HSA binding ability. It was previously reported that the conformational change of serum albumin occurring at pH = 3.0 effectively cause dissociation of bound L-tryptophan molecules [32]. 13 Further characterizations of HSA unfolding in the acid denatured state showed that a significant amount of the secondary structure was maintained, while a significant amount of the tertiary structure was lost [50]. This structural change may provide an opportunity for HSA regeneration after D,L-tryptophan separation. Nevertheless, the reusability of HSA depends on the reversibility of the HSA structure after acid denaturation. 1.5 Formation of hollow fiber membranes by phase inversion Compared to the flat sheet membrane configuration, the hollow fiber configuration offers several advantages, namely a high membrane area per unit volume of membrane module, self-support ability, flexibility and ease of handling during module preparation and operation [51]. Therefore, the hollow fiber membrane configuration is preferable for membrane-based separations. Generally, the hollow fiber membrane prepared from the polymeric material is fabricated through the phase inversion process. The phase inversion of hollow fiber membrane is more complex than the flat sheet as it involves more parameters to alter the morphology of the final membrane [52]. The hollow fiber spinning process is schematized in Fig. 1.4. 14 Figure 1.4 Schematic of hollow fiber spinning [53] After being extruded from the spinneret, the internal surface of the nascent fiber is in direct contact with the bore fluid and thus, the internal coagulation starts to occur. The partial coagulation of the membrane outer surface starts at the air gap region as a result of the air relative humidity. The external coagulation is completed once the nascent fiber reaches the external coagulation bath. The inner and outer morphologies of membrane surface are governed by the chemistry of the bore fluid and the external coagulant, respectively. Water is often selected as the external coagulant to lower down the production cost and being an environmentally benign. Macrovoids have been considered as an undesirable structure irregularity due to its weaker mechanical strength which may cause membrane failure at high hydraulic pressure. Several methods were proposed to mitigate the macrovoids formation during phase inversion: (1) increasing the polymer dope viscosity [54], (2) increasing the extrusion rate of polymer dope and thus, increasing shear rate [55], (3) inducing a 15 delayed demixing [56], (4) adding surfactants [57], (5) adding viscosity-enhancer additives [58], and (6) reducing the air gap distance [59]. 1.6 Research objectives Despite of all the advantages possessed by the affinity UF system, its commercial application for enantioseparation is limited by the recovery and reusability of the stereoselective ligands. In this respect, the albumin particularly from human serum (HSA) has shown potencies due to the impermanent nature of the association with the enantiomer. Though during earlier investigation on the enantioseparation of tryptophan with BSA as a stereoselective ligand, it was revealed that the BSA undergoes conformational change at pH = 3.0 releasing the bound L-tryptophan molecules [32]. Nevertheless, further investigations are still needed to verify the conformational and binding reversibility of albumin after being denatured by acid. Therefore, the objective of this research is to investigate the regeneration and reusability of stereoselective ligands by utilizing both renatured and recovered HSA in the separation experiment of D,L-tryptophan racemic mixture. Moreover, this research is also aim to develop hollow fiber membrane with low-fouling property and appropriate pore size for complete retention of the enantiomer-ligand complex and thus, ensuring high recovery of the steroselective ligand for subsequent separations. 16 Chapter 2. Experimental 2.1 Materials A commercial polyethersulfone (PES) was chosen as the polymeric material to fabricate the hollow fiber membranes. PES is preferred in this study because of its good thermal and chemical resistances, wide pH tolerance, ease of fabrication, and relatively low price [60]. A commercial Radel® A PES was obtained from Solvay Advanced Polymers L.L.C and N-Methyl-2-pyrrolidone (NMP) as a solvent was supplied by Merck. Figure 2.1 Chemical structure of PES polymer The application of PES membranes in biomolecules separation is limited by its hydrophobic property and thus, a rapid progressivity of membrane fouling is normally observed. Several surface modification methods were proposed to alter the membrane hydrophilicity, such as plasma treatment [61], grafting with hydrophilic monomers [62], sulfonation reaction [63], and direct blending with hydrophilic additives [64]. In this research, polyvinylpyrrolidone (PVP) with an average molecular weight of 360 kDa purchased from Merck was selected as an additive. PVP is a water soluble material that is normally applied in the biomedical applications. In membrane fabrication, blending of PVP with hydrophobic polymers to increase the hydrophilicity of the resultant membrane has been reported [65-66]. In addition, PVP 17 is also functioned as a pore-former due to the feasibility of the unblended PVP to be removed from the membrane matrix. Figure 2.2 Chemical structure of PVP Sodium hypochlorite (NaOCl) from Acros and glycerol from Merck were used as the post-treatment agents for the hollow fiber membranes generated in this work. Polyethyleneglycol (PEG) from Merck with molecular weights of 1,000, 4,000, 6,000 and 10,000 Da was used as a neutral solute for characterizations of pore size and pore size distribution of hollow fiber membranes. Disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), sodium hydroxide (NaOH) and phosphoric acid (H3PO4) were purchased from Merck for the preparation of buffer solutions. The racemic mixture of D,L-tryptophan (99%) was purchased from Alfa Aesar. Human serum albumin from Sigma-Aldrich (96-99%) was used as a stereoselective ligand. α-Cyclodextrine (α-CD) as a background electrolyte was purchased from CycloLab. All of these chemicals were of reagent grade and used as received. 2.2 Dope preparation Both PES granules and PVP powders were dried at 120 ºC overnight under vacuum. The dried PES and PVP were then dissolved into a chilled NMP solvent under a 18 vigorous stirring until the homogeneous and viscous polymer dope was attained. Afterwards, the polymer dope was degassed overnight before spinning. 2.3 Hollow fiber spinning The hollow fiber membranes were spun through the spinning line depicted schematically in Fig. 2.3. Figure 2.3 Schematic diagram of hollow fiber spinning line The bore fluid and polymer dope were pumped at different flow rates depending on the experimental conditions from the syringe pumps through the inner and outer channels of the spinneret, respectively. After being discharge from the spinneret, both solutions passed through the air gap region and entered the coagulant bath. The nascent fibers were collected at the take-up drum rotating at a predetermined rate. Subsequently, the as-spun hollow fibers were immersed in a water bath for several days to let the solvent exchange process occur. The clean fibers were then immersed in an 8000 ppm sodium hypochlorite aqueous solution under stirring for 24 h in order 19 to remove the immiscible PVP from the membranes matrix. Afterwards, two different post-treatment procedures were performed: (1) soaking the membrane into a glycerol aqueous solution for the permeation and separation experiments and (2) freeze-drying the membrane for characterizations. 2.4 Morphology study by FESEM The morphology of hollow fiber membranes was observed by the field emission scanning electron microscopy (FESEM) on a JEOL JSM-6700F. The samples were prepared by immersing the fiber in liquid nitrogen, fracturing and then coating with platinum using a JEOL JFC-1300 coater. 2.5 Contact angle measurements The membrane contact angle was measured by a KSV Sigma 701 Tensiometer from KSV Instruments Limited. The sample was brought into contact with deionized (DI) water and the force was recorded simultaneously. The contact angle can then be calculated by the software upon inputting the sample geometry and the water surface tension. For each condition, the average contact angle value obtained from three samples was reported. 2.6 Module fabrications The membrane modules were prepared by bundling 10 fibers for each module with a Teflon tape at both ends. The fiber bundle was inserted into a tubing (15 cm length) connected with two fittings. Afterward, the gap between the fiber bundle and the fitting was filled with the cotton before sealing with the epoxy. The similar procedure 20 was carried out for the other side of the module after the epoxy was completely hardened. 2.7 PWP and pore size distribution characterizations Each module was rinsed with DI water to remove the glycerol before every characterization and separation experiments. The pure water permeability (PWP) and pore size distribution characterizations were carried out at 15 °C and 2 bar pressure. The experimental setup applied for both measurements are illustrated in Fig. 2.4. A = Feed tank, B = Cooling coil, C = Filter, D = High pressure pump, E = Pulsation damper, F = Bypass valve, G = Pressure gauge, H = Membrane module, I = Sampling valve, J = Rotameter Figure 2.4 Schematic diagram of self-designed UF setup for PWP and pore size distribution characterizations [67] For the characterization of pore size distribution, PEG solutions with different molecular weights (MW) were prepared for the solute rejection (RT) test. The Stokes radius (rs) of solutes with different MW was calculated using the following equation: log rs = −1.32 + 0.395log MW (2.1) 21 The solute rejection (RT) can be expressed by a log-normal probability function of solute size [68] as shown in the following equation: cp 1 1 = RT =− erf ( y ) = cf 2π y= ln rs − ln µ s ln σ g y ∫e − ( u 2 /2) du (2.2) −∞ (2.3) where cp and cf are the permeate and feed concentrations, respectively, µs is the geometric mean solute radius at RT = 50%, rs is the solute radius, σg is the ratio between rs at RT = 84.13% to that at RT = 50%. By assuming the mean pore radius (µp) and the standard deviation (σp) are equal to µs and σg, respectively, the membrane pore size distribution can be expressed in the probability density function derived from Eq. (2) [69]: ( ln r − ln µ )2 dRT ( rp ) 1 p p exp − = 2 drp rp ln σ p 2π 2 ( ln σ p ) (2.4) where rp is the membrane pore radius. 2.8 Tryptophan separation performance The first step was to prepare the racemic solution with a concentration of 0.1 mM by dissolving D,L-tryptophan powders in a 10 mM phosphate buffer adjusted to pH 9.0. HSA was then added to the racemic solution to achieve a molar ratio of 2:1 of HSA to tryptophan. Upon addition of HSA, there was a shift in solution pH and hence, sodium hydroxide was added to adjust the pH back to 9.0. The resulting solution was stirred for 3 h to reach the binding equilibrium. Thirdly, the HSA-tryptophan mixture solution was fed into the self-designed UF setup at 15 °C and 0.5 bar for the separation experiments. An overall-recycle mode where both retentate and permeate 22 solutions were recycled back to the feed tank was carried out to keep the concentration of the feed solution constant. During the experimental running, the permeate samples were collected at regular intervals and were analyzed using a P/ACETM MDQ capillary electrophoresis (CE) system from Beckman Coulter. Unless otherwise stated, the data were acquired using a 50 cm long fused silica capillary with an inner diameter of 75 µm from Agilent Technologies. The instantaneous separation factor (α) and the instantaneous yields of D-tryptophan and L-tryptophan (YD and YL) were calculated using the following equations: α= Cp,D ⁄Cf,D YD = YL = Cp,L ⁄Cf,L Cp,D Cf,D Cp,L Cf,L X 100% X 100% (2.5) (2.6) (2.7) where Cp,D and Cp,L are the concentrations of D-enantiomer and L-enantiomer in the permeate side, respectively, while Cf,D and Cf,L are the concentrations of D-enantiomer and L-enantiomer in the feed side, respectively. Both Cf,D and Cf,L were acquired before the addition of HSA powders. 2.9 HSA denaturation and renaturation study A series of experiments were designed to explore the evolution of separation factor of HSA as a function of denaturation-renaturation times and to determine the percentage of HSA recovered at the end of each cycle mode. Each cycle mode is an independent and parallel experiment as illustrated in Fig. 2.5. It could be seen that different cycle modes mean the different times of acidification and basification process before the 23 HSA-tryptophan mixture solution was fed into the UF setup for separation experiments. Figure 2.5 Schematic of HSA denaturation and renaturation experiments In the HSA denaturation and renaturation study, the separation experiment consisted of two parts, namely permeation part and recovery part as shown in Fig. 2.6. A continuous diafiltration mode was conducted at 15 °C and 0.5 bar to recover the HSA from the feed solution. Herein, only the retentate solution was recycled back to the feed tank and the fresh buffer was topped up into the feed tank to balance the loss of the permeate solution [70]. 24 Figure 2.6 Schematic of tryptophan separation experiments for HSA denaturation and renaturation study under diafiltration mode The permeation part was run for 4 h and the amounts of D-tryptophan and L- tryptophan collected at the permeate container were analyzed using the CE system. The overall separation factor (αo) and the overall yield of L-tryptophan (YL,o) were calculated using the following equation: αo = YL,o = np,D ⁄nf,D np,L ⁄nf,L np,L nf,L (2.8) X 100% (2.9) where np,D and np,L are the moles of D-enantiomer and L-enantiomer in the collected permeate solution, respectively, at the end of the permeation part, while nf,D and nf,L are the initial moles of D-enantiomer and L-enantiomer in the feed solution, respectively. Both nf,D and nf,L were acquired before the addition of HSA powders. As shown in Fig. 2.6, the solution retained in the feed tank in the permeation part was used as a feed in the recovery part after reducing its pH value to 3.0 via the addition 25 of phosphoric acid. The same UF system and operating parameters were used as the permeation part with a running time of 5 h. At the end of each cycle mode, the HSA content in the retained solutions was analyzed with the CE system using a 50 cm long neutral polyacrylamide coated capillary with an inner diameter of 50 µm from Beckman Coulter. The recovery percentage of HSA (% Rec,HSA) was calculated using the following equation: % Rec,HSA = Cr,HSA Cf,HSA x 100% (2.10) where Cr,HSA and Cf,HSA are the concentrations of HSA at the retentate and feed sides, respectively. 2.10 HSA regeneration and reusability Fig. 2.6 can also be used to explore the reusability of the recovered HSA as a stereoselective ligand in the real case which included two steps of separation experiments, and the test was accomplished within two consecutive days. The first step was exactly the same as the 1-cycle mode in the HSA denaturation-renaturation experiment, whereas, in the second step, the HSA recovered from the first step was reused as a stereoselective ligand to separate the second batch of fresh tryptophan racemic mixture. 2.11 CD measurements of HSA To characterize the structure of HSA in solutions with various pH values, Circular Dichroism (CD) measurements were conducted on a spectropolarimeter Jasco J-810. For all samples, the temperature was maintained at 15 ºC and the average result of 26 three scans was reported. The native HSA sample was prepared by dissolving HSA powders in a 10 mM phosphate buffer with a pH value of 9.0 to achieve a concentration of 0.75 g/l and 5 g/l for the far-UV CD and near-UV CD, respectively. Upon addition of HSA, there was a shift in solution pH and hence, sodium hydroxide was added to adjust the pH back to 9.0. The acid-denatured HSA sample was then prepared by adding phosphoric acid into the native HSA solution to reduce the pH value to 3.0 and afterwards, the base-renatured HSA sample was attained by adding sodium hydroxide to readjust the pH value back to 9.0. The ionic strength of the HSA samples was regulated by a dialysis membrane prior to CD measurements. The 1 mm pathlength cell was used for far-UV CD, while the 5 mm pathlength cell was used for near-UV CD. The buffer interference was eliminated by subtracting the buffer spectrum from the obtained sample spectra. The mean residue ellipticity (MRE, deg.cm2/dmol) was calculated using the following equation [71]: MRE = θobs (2.11) 10 x CHSA x n x l where 𝜃obs is the CD measurement (mdeg), n is the number of amino acid residues, l is the cell pathlength (cm) and CHSA is the HSA concentration (M). The MRE obtained at the wavelength of 222 nm was further used to calculate the α-helical content using the following equation [71]: MRE222 −2340 % helix = � 30300 � X 100% (2.12) 27 Chapter 3. Exploration of Regeneration and Reusability of HSA as a Stereoselective Ligand for Chiral Separation in Affinity UF 3.1 Characterizations of hollow fiber membranes The detailed dope compositions and spinning parameters for fabrication of hollow fiber membranes are summarized in Table 3.1. Table 3.1 Spinning parameters for hollow fiber membranes ID Dope composition Fiber-4.5cm Fiber-10cm Fiber-15cm PES/PVP/NMP (20/10/70 wt%) Bore fluid composition NMP/water (86/14 wt%) Dope flow rate (ml/min) 2 Bore fluid flow rate (ml/min) Air gap (cm) 0.8 4.5 10 Take up rate (cm/min) 1240 External coagulant Water Spinning temperature (°C) 25 Coagulation bath temperature (°C) 25 15 The addition of PVP into the polymer dope mainly serves three purposes in this research. The first is to enhance the dope viscosity and thus retard the formation of macrovoids, which has been proven by the cross-sectional FESEM images of hollow fibers (Fig. 3.1). The second is to increase the membrane hydrophilicity [65-66] and consequently reduce the non-specific adsorption of the HSA and tryptophan molecules within the membrane which is very helpful to improve the HSA recovery. The third is to function as a pore forming agent due to the feasibility of the unblended PVP to be removed from the membrane matrix by the NaOCl treatment [72]. 28 Figure 3.1 Comparison of FESEM images of PES hollow fiber membranes spun at various air gap distances The pure water permeability, mean pore radius, geometric standard deviation and molecular weight cut-off (MWCO) of hollow fiber membranes spun at different air gap distances are given in Table 3.2, while the cumulative pore size distribution and probability density function curves are illustrated in Fig. 3.2. Table 3.2 Comparison of PWP and parameters from neutral solute rejection experiments PWP Mean pore radius MWCO Standard deviation 2 (nm) (kDa) (L/(m bar h)) Fiber-4.5cm 74.79 + 4.85 3.1 + 0.03 1.68 + 0.01 10.92 + 0.17 Fiber-10cm 105.37 + 2.46 3.43 + 0.09 1.66 + 0.04 13.28 + 0.51 Fiber-15cm 116.27 + 2.72 4.06 + 0.17 1.75 + 0.08 20.23 + 1.03 29 Figure 3.2 Cumulative pore size distribution (A) and probability density function (B) curves for hollow fiber membranes It can be seen in Table 3.2 that the PWP value increases as an order of fiber-4.5cm < fiber-10cm < fiber-15cm. This change is probably due to the increment in the elongational stress induced by gravity as the air gap distance increases. After being extruded from the spinneret, the outer surface of the nascent fibers contacts with the air and the moisture-induced phase separation occurs. Afterwards, a high elongational stress may draw polymer chains of the fibers at the initial stage of phase separation and hence, induce the formation of pores that is confirmed by the increment in membrane MWCO (Table 3.2) and the shifting in the pore size distribution curves (Fig. 3.2) with increasing air gap distance. A high rejection of HSA is one of prerequisites to ensure a high separation factor of the tryptophan racemic mixture in the affinity UF system. CE analyses of permeate solutions through three types of PES membranes developed in this study indicate that all membranes can fully retain HSA as shown by the capillary electropherograms in Fig. 3.3. 30 D-tryptophan L-tryptophan Figure 3.3 Capillary electropherograms of permeate samples via hollow fiber membranes 3.2 Tryptophan separation of hollow fiber membranes A comparison of tryptophan separation factor among these three types of membranes is illustrated in Fig. 3.4. It can be seen that despite the difference in pore size distribution, all of them exhibit a similar separation performance with time. This implies that as long as the complete HSA rejection is attained, the separation factor is determined dominantly by the intrinsic association constants of HSA with two enantiomers, instead of the membrane pore size. 31 Figure 3.4 Comparison of tryptophan separation factor of hollow fiber membranes This point seems contradictory with the previous work reported by our group that suggested a suitable membrane pore size contributed to an enhanced separation performance [40]. This may be due to the difference in operation modes in these two works. The dead-end mode was used in the previous work, whereas the cross-flow mode where the feed solution flowed tangent to the membranes surface was used in the current work. Together with the addition of hydrophilic PVP and smaller membrane pore size (i.e. MWCO 19 kDa in the current work vs. 30 kDa in the previous work), the probability to retain the HSA molecules within the membrane cross-section is significantly reduced in this work and therefore, it may not provide a second-stage binding opportunity for L-tryptophan as shown in the previous work. Based on the above results, fiber-15cm was therefore selected as a specimen to explore the feasibility of HSA regeneration and reusability in the next section. 32 3.3 Tryptophan separation of native and renatured HSA Comparisons of tryptophan separation of native and renatured HSA as well as HSA recovery as a function of denaturation-renaturation times are summarized in Table 3.3. Table 3.3 Overall separation factor, overall L-tryptophan yield and percentage of recovered HSA for fiber-15cm Overall separation Overall L-tryptophan yield Percentage of recovered factor (αo) (YL,o) HSA (% Rec,HSA) 1-cycle 5.29 + 0.28 10.62 + 0.08 87.62 + 4.42 2-cycle 4.58 + 0.27 11.33 + 0.22 90.12 + 4.19 3-cycle 4.49 + 0.3 11.84 + 0.34 83.88 + 3.56 4-cycle 3.3 + 0.2 21.5 + 0.42 97.18 + 4.52 5-cyclea - - - a The data could not be attained due to the HSA precipitation induced by a high salt concentration after several times of denaturation-renaturation process. It can be seen in Table 3.3 that after three times of denaturation-renaturation process are carried out in the 4-cycle mode, the overall tryptophan separation factor is > 3.0. This implies that by simply readjusting the medium pH value, the capability of HSA to selectively bind L-tryptophan over D-tryptophan can be recovered. Nevertheless, the overall separation factor decreases with increasing times of denaturation-renaturation process. There are two reasons that might be responsible for the decline in overall separation factors. One is that the acidic condition may induce an irreversible structural change of HSA, and the other is that the salt concentration in the HSAtryptophan mixture solution is enhanced by the continuous addition of phosphoric acid and sodium hydroxide. 33 The CD technique was chosen to examine the structure of the native, acid-denatured and base-renatured HSA samples. The far-UV CD and near-UV CD spectra of these samples are shown in Fig. 3.5 A and Fig. 3.5 B, respectively. Since the HSA spectra after two or more times of denaturation-renaturation process are similar to those after one time, only the HSA spectra after one time are plotted. Figure 3.5 Far-UV CD spectra (A) and near-UV CD spectra (B) for native, one time acid-denatured and one time base-renatured HSA It can be seen in Fig. 3.5 A that the far-UV CD spectra for all HSA samples are similar yet with different intensities, and show two minima at 208 nm and 222 nm which indicate an α-helical structure. The MRE values obtained at the wavelength of 222 and 262 nm as well as the calculated α-helical contents of the native, aciddenatured and base-renatured HSA samples are summarized in Table 3.4. 34 Table 3.4 Summary of far-UV CD and near-UV CD data for native, acid-denatured and base-renatured HSA with different denaturation-renaturation times MRE at 222 nm MRE at 262 nm % helix (deg cm2/dmol) (deg cm2/dmol) Native HSA 21,095 + 655 61.9 + 2.16 144.07 + 4.26 1 time base-renatured 20,729 + 388 60.69 + 1.28 130.48 + 4.48 2 time base-renatured 19,823 + 614 57.7 + 2.03 126.25 + 7.84 3 time base renatured 19,350 + 520 56.14 + 1.71 128.46 + 5.31 4 time base-renatured 18,329 + 493 52.77 + 1.6 119.89 + 4.96 Acid-denatured HSA 14,259 + 383 39.34 + 1.19 110.05 + 4.55 As shown in Table 3.4, the α-helical content of the native HSA is around 61%, while the acid-denatured HSA exhibits a much lower value suggesting that under the acidic condition, the structural change of HSA happens. It has been reported that in the acidic condition, hydrophobic forces, disulfide bonds, salt bridges and metal ionprotein interactions that favor protein folding may fail to overcome the intramolecular charge repulsion that favors protein unfolding [73]. Another point worthy of notice in Fig. 3.5 A is that after adding the base to the pH value of 9.0, the acid-denatured HSA is able to refold to gain almost all of the secondary structures. Nevertheless, the αhelical content in Table 3.4 shows a declining trend with increasing denaturationrenaturation times. The near-UV CD is a useful technique to get insight of the tertiary structure of proteins [74]. It can be seen in Fig. 3.5 B that the near-UV CD spectra of the native, acid-denatured and base-renatured HSA samples are similar yet with different intensities, and show two minima at 262 nm and 268 nm which are characteristic of disulfide bond [75]. Similarly, the acid-denatured HSA with a much lower ellipticity value in Table 3.4 is able to refold to gain most of the tertiary structures after the 35 medium pH is readjusted back to pH 9.0. However, the ellipticity value of the baserenatured HSA is lower than its native counterpart and declines with increasing denaturation-renaturation times, which imply that more of the tertiary structures may lose after more times of acidification. Both of far-UV CD and near-UV CD spectra suggest that the structural change of HSA might occur after acidification and basification, and its extent becomes more prominent with increasing denaturationrenaturation times. Other than the structural change, the decline in overall separation factors with increasing denaturation-renaturation times in Table 3.4 is perhaps also due to the accumulation of buffer salts that leads to an increase in the solution ionic strength. The ionic strength promotes the electrolyte shielding, thus reducing the electrostatic attraction between the HSA and L-tryptophan. The similar phenomenon has been reported in a high performance liquid chromatography (HPLC) system where HSA was immobilized onto the HPLC column [76]. Moreover, the HSA binding cavity becomes less accessible with increasing ionic strength. This is because a higher ionic strength raises the surface tension that leads to an increase in hydrophobic interaction of nonpolar groups inside the HSA binding cavity and subsequently, a decrease in radii of the binding cavity [41]. As a result, both effects of ionic strength may reduce the amount of L-tryptophan molecules bound to HSA, hence causing more L- tryptophan molecules to appear at the permeate side of membranes as shown in Table 3.4. In addition, Table 3.4 displays a high recovery percentage of HSA of above 80% regardless of denaturation-renaturation times, which is very helpful to provide a 36 sufficient source for the following experiment of HSA reusability. The high recovery of HSA may be a synergistic effect of two factors. One is the suitable pore size of hollow fiber membranes developed in this work that prevents HSA molecules from entering and passing through the membrane. HSA has an approximate dimension of 80 x 80 x 30 Å [77], while the membrane average pore sizes are in the range of 30-40 Å (Table 3.2). The other is the improved hydrophilicity of membranes after the addition of PVP. The contact angle of PES/PVP membranes spun in this work is 63.0o ± 1.1o, while the contact angle of neat PES membranes spun with the very similar polymer concentration (i.e. 21 wt%) in our group is 71.2o ± 1.7o. The improvement in hydrophilicity can significantly suppress the hydrophobic adsorption of HSA on the membrane surface, thus reducing membrane fouling. 3.4 Tryptophan separation of native and recovered HSA Through the acid-denaturation followed by the base-renaturation, the feasibility of the renatured HSA as a stereoselective ligand for D,L-tryptophan separation has been demonstrated. However, it is more attractive for industrial applications to explore the feasibility of the HSA recovered from the previous separation experiment to be utilized as a stereoselective ligand in the subsequent separation experiment. Therefore, L-tryptophan and D-tryptophan yields between the native and recovered HSA are compared as a function of time in Fig. 3.6. It can be seen from Fig. 3.6 A that the yield of L-tryptophan is similar between both cases. On the other hand, the D- tryptophan yield of the recovered HSA is higher than the native HSA as shown in Fig. 3.6 B which may be due to the saturation of the non-stereospecific binding sites of HSA by the D-tryptophan molecules during the previous separation experiment. 37 Consequently, the separation factor of the recovered HSA is slightly higher than the native HSA as shown in Fig. 3.7. Figure 3.6 Comparison of yields of L-tryptophan (A) and D-tryptophan (B) between native (●) and recovered (○) HSA Figure 3.7 Comparison of tryptophan separation factor between native (●) and recovered (○) HSA Nevertheless, on the other hand, this result seems contradictory with the previous result in which the separation factor decreases with increasing denaturationrenaturation times in Table 3.3. This discrepancy may arise from the fact that the solution ionic strength is different between these two experiments. A lesser amount of 38 buffer salts was retained in the first separation step of the HSA reusability experiment since during the HSA recovery after separation of the first batch of D,L-tryptophan, the acidic buffer solution was diluted by the top up buffer and most of the formed buffer salts were also transferred into the membrane permeate side. The difference between the denaturation-renaturation and reusability experiments may indirectly give a hint that the increment in solution ionic strength may be more responsible than the structural change of HSA for the decline in separation factors in the denaturationrenaturation experiment. 39 Chapter 4. Conclusion The following conclusions can be drawn from this work: 1. In the affinity UF system operated with a cross-flow mode, the membrane pore sizes evaluated here did not affect the tryptophan separation performance provided that the complete rejection of HSA as a stereoselective ligand is attained. 2. The binding capability of the acid-denatured HSA to L-tryptophan is recovered through the basification process. 3. The tryptophan separation performance of the renatured HSA as a stereoselective ligand diminishes with increasing denaturation-renaturation times. Besides the irreversible structural change of HSA molecules caused by the acidification process, the increment in solution ionic strength may be a more responsible factor. A higher solution ionic strength may lead to a decrease in electrostatic attraction between HSA and L-tryptophan via electrolyte shielding and meanwhile, reduce the accessibility of the HSA binding cavity to L-tryptophan because of the shrinkage of the binding cavity area. 4. A high recovery of HSA is achieved through tuning the membrane pore size and incorporating the hydrophilic PVP additives, which provides a good base for the study of HSA reusability. The native and recovered HSA samples exhibit a similar separation performance in this work, which demonstrates the reusability of HSA in reality and is very inspiring for the large-scale application of the affinity UF system in chiral separation. 40 Bibliography [1] H.Y. Aboul-Enein, I. Ali, Chiral Separations By Liquid Chromatography And Related Technologies, CRC Press, New York, 2003 [2] E.H. Easson, E. Stedman, Studies on the relationship between chemical constitution and physiological action, Biochem. J. 27 (1933) 1257-1266 [3] V. Gunzler, Thalidomide in human immunodeficiency virus (HIV) patients. A review of safety considerations, Drug Saf. 7 (1992) 116-134 [4] U.S. Food and Drug Administration, FDA’s policy statement for the development of new stereoisomeric drugs, Chirality 4 (1992) 338-340 [5] H. Caner, E. Groner, L. Levy, I. Agranat, Trends in the development of chiral drugs, Drug Discovery Today 9 (2004)105-110 [6] J.E. Rekoske, Chiral separations, AIChE Journal 47 (2001) 2-5 [7] N.M. Maier, P. Franco, W. Lindner, Separation of enantiomers: needs, challenges, perspectives, J. Chromatography A 906 (2001) 3-33 [8] S. Beilles, P. Cardinael, E, Ndzie, S. Petit, G. Coquerel, Preferential crystallisation and comparative crystal growth study between pure enantiomer and racemic mixture of a chiral molecule: 5-ethyl-5-methylhydantoin, Chem. Eng. Sci. 56 (2001) 22812294 [9] A.A. Rodrigo, H. Lorenz, A. Seidel-Morgenstern, Online monitoring of preferential crystallization of enantiomers, Chirality 16 (2004) 499-508 [10] E.J. Valente, T.N. Smith, M.E. Harris, Discrimination in resolving systems. VI. Comparison of the diastereomers of deoxyephedrinium and ephedrinium 4 ′ fluoromandelates, Chirality 13 (2001) 244-250 [11] J.G. Deng, Y.X. Chi, F.M. Fu, X. Cui, K.B. Yu, J. Zhu, Y.Z. Jiang, Resolution of omeprazole by inclusion complexation with a chiral host BINOL, Tetrahedron: Asymmetry 11 (2000) 1729-1732 [12] H. Pellissier, Dynamic kinetic resolution, Tetrahedron 59 (2003) 8291-8327 [13] M. Tokunaga, J.F. Larrow, F. Kakiuchi, E.N. Jacobsen, Asymmetric catalysis with water: efficient kinetic resolution of terminal epoxides by means of catalytic hydrolysis, Science 277 (1997) 936-938 [14] U.T. Strauss, K. Faber, Deracemization of (±)-mandelic acid using a lipase– mandelate racemase two-enzyme system, Tetrahedron: Asymmetry 10 (1999) 40794081 41 [15] H.E. Schoemaker, D. Mink, M.G. Wubbolts, Dispelling the Myths--Biocatalysis in Industrial Synthesis, Science 299 (2003) 1694-1697 [16] K.L. Williams, L.C. Sander, Enantiomer separations on chiral stationary phases in supercritical fluid chromatography, J. Chromatogr. A. 785 (1997) 149-158 [17] M. Schulte, J. Strube, Preparative enantioseparation by simulated moving bed chromatography, J. Chromatogr. A. 906 (2001) 399-416 [18] E.R. Francotte, Enantioselective chromatography as a powerful alternative for the preparation of drug enantiomers, J. Chromatogr. A. 906 (2001) 379-397 [19] R. Xue, L.Y. Chu, J.G. Deng, Membranes and membrane processes for chiral resolution, Chem. Soc. Rev. 37 (2008) 1243-1263 [20] C.A.M. Afonso, J.G. Crespo, Recent advances in chiral resolution through membrane-based approach, Angew. Chem. Int. Ed. 42 (2004) 5293-5295 [21] J.H. Kim, J.H. Kim, J. Jegal, K. Lee, Optical resolution of -amino acids through enantioselective polymeric membranes based on polysaccharides, J. Memb. Sci. 213 (2003) 273-283 [22] S. Xie, W. Wang, P. Ai, M. Yang, L. Yuan, Chiral separation of (R,S)-2-phenyl1-propanol through cellulose acetate butyrate membranes, J. Memb. Sci. 321 (2008) 293-298 [23] J. Wang, C.J. Fu, T. Lin, L.X. Yu, S.L. Zhu, Preparation of chiral selective membranes for electrodialysis separation of racemic mixture, J. Memb. Sci. 276 (2006) 193-198 [24] N. H. Lee, C. W. Frank, Separation of chiral molecules using polypeptidemodified poly(vinylidene fluoride) membranes, Polymer 43 (2002) 6255-6262 [25] M. Yoshikawa, K. Murakoshi, T. Kogita, K. Hanaoka, M.D. Guiver, G.P. Robertson, Chiral separation membranes from modified polysulfone having myrtenalderived terpenoid side groups, Europ. Polym. J. 42 (2006) 2532–2539 [26] H.M. Krieg, J. Lotter, K. Keizer, J.C. Breytenbach, Enrichment of chlorthalidone enantiomers by an aqueous bulk liquid membrane containing β-cyclodextrin, J. Memb. Sci. 167 (2000) 33-45 [27] P. Dzygiel, P. Wieczorek, Extraction of amino acids with emulsion liquid membranes using industrial surfactants and lecithin as stabilizers, J. Memb. Sci. 172 (2000) 223-232 [28] K. Sakaki, S. Hara, N. Itoh, Optical resolution of racemic 2-hydroxy octanoic acid using biphasic enzyme membrane reactor, Desalination 149 (2002) 247-252 42 [29] A. Bodalo, J.L. Gomez, E. Gomez, M.F. Maximo, M.C. Montiel, Study of laminoacylase deactivation in an ultrafiltration membrane reactor, Enzyme Microb. Technol. 35 (2004) 261-266 [30] J.T.F. Keurentjes, L.J.W.M. Nabuurs, E.A. Vegter, Liquid membrane technology for the separation of racemic mixtures, J. Memb. Sci. 113 (1996) 351-360 [31] J.L. Lopez, S.L. Matson, A multiphase/extractive enzyme membrane reactor for production of diltiazem chiral intermediate, J. Memb. Sci. 125 (1997) 189-211 [32] A. Higuchi, Y. Ishida, T. Nakagawa, Surface modified polysulfone membranes: separation of mixed proteins and optical resolution of tryptophan, Desalination 90 (1993) 127-136 [33] S. Poncet, J. Randon, J.L. Rocca, Enantiomeric separation of tryptophan by ultrafiltration using the BSA solution system, Separation Sci. Tech. 32 (1997) 20292038 [34] J. Romero, A.L. Zydney, Chiral separations using ultrafiltration with a stereoselective binding agent, Separation Purif. Tech. 36 (2001) 1575-1594 [35] J. Romero, A.L. Zydney, pH and salt effects on chiral separation using affinity ultrafiltration, Desalination 148 (2002) 159-164 [36] J. Romero, A.L. Zydney, Staging of affinity ultrafiltration processes for chiral separations, J. Membr. Sci. 209 (2002) 107-119 [37] A. Yudiarto, E. Dewi, T. Kokugan, Separation of isomers by ultrafiltration using modified cyclodextrins, Separation Purif. Tech 19 (2000) 103-112 [38] A. Higuchi, H. Yomogita, B.O. Yoon, T. Kojima, M. Hara, S. Maniwa, M. Saitoh, Optical resolution of amino acid by ultrafiltration using recognition sites of DNA, J. Membr. Sci. 205 (2002) 203-212 [39] T.J.M. De Bruin, A.T.M. Marcelis, H. Zuilhof, L.M. Rodenburg, H.A.G. Niederlander, A. Koudijs, P.E.M. Overdevest, A. Van Der Padt, E.J.R. Sudholter, Separation of amino acid enantiomers by micelle-enhanced ultrafiltration, Chirality 12 (2000) 627-636 [40] H. Wang, Y. Li, T.S. Chung, A fine match between the stereoselective ligands and membrane pore size for enhanced chiral separation, AIChE J. 55 (2009) 22842291 [41] E. Peyrin, Y.C. Guillaume, C. Guinchard, Characterization of solute binding at human serum albumin site II and its geometry using a biochromatographic approach, Biophys. J. 77 (1999) 1206-1212 [42] U. Kragh-Hansen, Molecular aspects of ligand binding to serum albumin, Pharm. Rev. 33 (1981) 17-53 43 [43] C. Lagercrantz, T. Larrson, I. Denfors, Stereoselective binding of the enantiomers of warfarin and tryptophan to serum albumin for some different species studied by affinity-chromatography on columns of immobilized serum-albumin, Comp. Biochem. Physiol. C. 69 (1981) 375-378 [44] K.J. Fehske, W.E. Muller, U. Wollert, The location of drug binding sites in human serum albumin, Biochem. Pharmacol. 30 (1981) 687-692 [45] G. Sudlow, D.J. Birkett, D.N.Wade, The characterization of two specific drug binding sites on human serum albumin, Mol. Pharmacol. 11 (1975) 824-832 [46] E. Peyrin, Y.C. Guillaume, N. Morin, C. Guinchard, Sucrose dependence of solute retention on human serum albumin stationary phase: hydrophobic effect and surface tension considerations, Anal. Chem. 70 (1998) 2773-3102 [47] E. Peyrin, Y.C. Guillaume, C. Guinchard, Peculiarities of dansyl amino acid enantioselectivity using human serum albumin as a chiral selector, J. Chromatogr. Sci. 36 (1998) 97-103 [48] E. Peyrin, Y.C. Guillaume, N. Morin, C. Guinchard, Retention behavior of D,Ldansyl-amino acids on a human serum albumin stationary phase: effect of a mobile phase modifier, J. Chromatogr. A. 808 (1998) 113-120 [49] E. Peyrin, Y.C. Guillaume, Chiral discrimination of N-(dansyl)-DL-amino acids on human serum albumin stationary phase: effect of a mobile phase modifier, Chromatographia. 48 (1998) 431-435 [50] S. Muzammil, Y. Kumar, S. Tayyab, Molten globule-like state of human serum albumin at low pH, Eur. J. Biochem. 266 (1999) 26-32 [51] T. Matsuura, Synthetic Membranes and Membrane Separation Process, CRC Press, Boca Raton, 1994 [52] T.S. Chung, The limitations of using Flory-Huggins equation for the states of solutions during asymmetric hollow fiber formation, J. Membr. Sci., 126 (1997) 19-34 [53] T.S. Chung, Fabrication of Hollow-Fiber Membranes by Phase Inversion, in: N.N. Li, A.G. Fane, W.S. Winston, T. Matsuura (Eds.), Advanced Membrane Technology and Applications, A John Wiley & Sons Inc., New Jersey, 2008 [54] T.S. Chung, E.R. Kafchinski, R. Vora, Development of a defect-free 6FDAdurene asymmetric hollow fiber and its composite hollow fibers, J. Memb. Sci. 88 (1994) 21-36 [55] J. Z. Ren, T.S. Chung, D.F. Li, R. Wang and Y. Liu, Development of asymmetric 6FDA-2,6 DAT hollow fiber membranes for CO2/CH4 separation - 1. The influence of dope composition and rheology on membrane morphology and separation performance, J. Membr. Sci. 207 (2002) 227-240 44 [56] J.H. Kim, B.R. Min, J. Won, H.C. Park, Y.S. Kang, Phase behavior and mechanism of membrane formation for polyimide/DMSO/water system, J. Membr. Sci. 187 (2001) 47-55 [57] H.A. Tsai, D.H. Huang, K.R. Lee, Y.C. Wang, C.L. Li, J. Huang, J.Y. Lai, Effect of surfactant addition on the morphology and pervaporation performance of asymmetric polysulfone membranes, J. Membr. Sci. 176 (2000) 97-103 [58] D.F. Li, T.S. Chung, R. Wang, Y. Liu, Fabrication of fluoropolyimide/polyethersulfone dual-layer asymmetric hollow fiber membranes for gas separation, J. Memb. Sci. 198 (2002) 211-223 [59] N. Widjojo, T.S. Chung, Thickness and air gap dependence of macrovoid evolution in phase-inversion asymmetric hollow fiber membranes, Ind. Eng. Chem. Res., 45 (2006) 7618-7626 [60] M. Cheryan, Ultrafiltration and Microfiltration Handbook, Technomic Publishing Co., Lancaster, PA, 1998 [61] D.S. Wavhal, E.R. Fisher, Membrane surface modification by plasma-induced polymerization of acrylamide for improved surface properties and reduced protein fouling, Langmuir 19 (2003) 79-85 [62] N. Hilal, L. Al-Khatib, B.P. Atkin, V. Kochkodan, N. Potapchenko, Photochemical modification of membrane surfaces for (bio)fouling reduction: a nanoscale study using AFM, Desalination 158 (2003) 65-72 [63] D. Lu, H. Zou, R. Guan, H. Dai, L. Lu, Sulfonation of polyethersulfone by chlorosulfonic acid, Polymer Bulletin 54 (2005) 16-19 [64] W. Zhao, Y. Su, C. Li, Q. Shi, X. Ning, Z. Jiang, Fabrication of antifouling polyethersulfone ultrafiltration membranes using Pluronic F127 as both surface modifier and pore-forming agent, J. Memb. Sci. 318 (2008) 405-412 [65] Q. Yang, T.S. Chung, M. Weber, Microscopic behavior of polyvinylpyrrolidone hydrophilizing agents on phase inversion polyethersulfone hollow fiber membranes for hemofiltration, J. Membr. Sci. 326 (2009) 322-331 [66] J.H. Kim, C.K. Kim, Ultrafiltration membranes prepared from blends of polyethersulfone and poly(1-vinylpyrrolidone-co-styrene) copolymers, J. Membr. Sci. 262 (2005) 60–68 [67] K.Y. Wang, T. Matsuura, T.S. Chung, W.F. Guo, The effects of flow angle and shear rate within the spinneret on the separation performance of poly(ethersulfone) (PES) ultrafiltration hollow fiber membranes, J. Membr. Sci. 240 (2004) 67-79 [68] B. Van der Bruggen, J. Schaep, D. Wilms, C. Vandecasteele, A comparison of models to describe the maximal retention of organic molecules in nanofiltration, Sep Sci Technol. 35 (2000) 169–182 45 [69] P. Aimar, M. Meireles, V. Sanchez, A contribution to the translation of retention curves into pore-size distributions for sieving membranes, J. Membr. Sci. 54 (1990) 321–338 [70] W.F. Blatt, S.M. Robinson, H.J. Bixler, Membrane ultrafiltration: the diafiltration technique and its application to microsolute exchange and binding phenomena, Anal. Biochem. 26 (1968) 151-173 [71] Y.H. Chen, J.T. Yang, H. Martinez, Determination of the secondary structure of proteins by circular dichroism and optical rotary dispersion, Biochemistry 11 (1972) 4120-4131 [72] Q. Yang, T.S. Chung, Y.E. Santoso, Tailoring pore size and pore size distribution of kidney dialysis hollow fiber membranes via dual-bath coagulation approach, J. Membr. Sci. 290 (2007) 153-163 [73] A.L. Fink, L.J. Calciano, Y. Goto, T. Kurotsu, D.R. Palleros, Classification of acid denaturation of proteins: intermediates and unfolded states, Biochemistry 33 (1994) 12504-12511 [74] S.M. Kelly, T.J. Ness, N.C. Price, How to study proteins by circular dichroism, Biochim. Biophys. Acta 1751 (2005) 119-139 [75] Y.L. Jin, M. Hirose, Partially folded state of the disulfide-reduced form of human serum albumin as an intermediate for reversible denaturation, J. Biol. Chem. 267 (1992) 14753-14758 [76] J. Yang, D.S. Hage, Role of binding capacity versus binding strength in the separation of chiral compounds on protein-based high-performance liquid chromatography columns Interactions of D- and L- tryptophan with human serum albumin, J. Chromatogr. A 725 (1996) 273-285 [77] S. Sugio, A. Kashima, S. Mochizuki, M. Noda, K. Kobayashi, Crystal structure of human serum albumin at 2.5 Å resolution, Protein Engineering 12 (1999) 439-446 46 Appendix List of papers published during M. Eng study 1. F. Edwie, Y. Li, T.S. Chung, Exploration of regeneration and reusability of human serum albumin as a stereoselective ligand for chiral separation in affinity ultrafiltration, Journal of Membrane Science 362 (2010) 501-508 2. F. Edwie, Y. Li, T.S. Chung, Application of membrane technologies for biomolecules separation by affinity binding to recovered stereospecific ligands, AIChE Annual Meeting (2011) Minneapolis, Minnesota 47 [...]... consisted of D-tryptophan inferring the preferential binding of L-tryptophan to BSA The separation factor of Dtryptophan over L-tryptophan as high as 7 was attained, which demonstrates the feasibility of an affinity UF system for chiral separation Interestingly, when the 9 solution pH was changed to 3.0, L-tryptophan was existed in the permeate solution indicating detachment of the L-tryptophan from... enantiomer binding with stereoselective ligands [30-36] The kinetic resolution is often catalyzed by enzymes, such as lipase to hydrolyze the enantiomers followed by membrane filtration to filter out the inhibitory byproducts The differences in the separation principle between enantioselective and nonenantioselective membranes are illustrated in Fig 1.2 Figure 1.2 Separation mechanisms of enantioselective and. .. size compared to the enantiomers and hence the enantiomer-ligand complex can be easily retained by the UF membrane, and (3) feasibility to be regenerated and reuse In most of the reported studies, tryptophan was commonly employed as the model enantiomers with various stereoselective ligands incorporated to achieved enantioseparation The examples of the commonly used stereoselective ligands are cyclodextrins... contribute to the HSA specificity and binding affinity [45] Later on, the binding chemistry of the site II of HSA in which the L-tryptophan is selectively bound was investigated through a series of studies by Peyrin et al [46-49] The binding mode was described in which the nonpolar residues inside the binding cavity of HSA were occupied by the hydrophobic groups of the bound molecule, while the cationic and. .. ligand, it was revealed that the BSA undergoes conformational change at pH = 3.0 releasing the bound L-tryptophan molecules [32] Nevertheless, further investigations are still needed to verify the conformational and binding reversibility of albumin after being denatured by acid Therefore, the objective of this research is to investigate the regeneration and reusability of stereoselective ligands by. .. where rp is the membrane pore radius 2.8 Tryptophan separation performance The first step was to prepare the racemic solution with a concentration of 0.1 mM by dissolving D,L-tryptophan powders in a 10 mM phosphate buffer adjusted to pH 9.0 HSA was then added to the racemic solution to achieve a molar ratio of 2:1 of HSA to tryptophan Upon addition of HSA, there was a shift in solution pH and hence, sodium... retentate solution was recycled back to the feed tank and the fresh buffer was topped up into the feed tank to balance the loss of the permeate solution [70] 24 Figure 2.6 Schematic of tryptophan separation experiments for HSA denaturation and renaturation study under diafiltration mode The permeation part was run for 4 h and the amounts of D-tryptophan and L- tryptophan collected at the permeate container... of HSA, it will not be attached to the stereospecific binding site of HSA Moreover, the HSA selectivity towards L-tryptophan is also dependent on the HSA conformation as the dimension and structure of the binding cavity may change as the HSA conformation is disrupted leading to a loss of the HSA binding ability It was previously reported that the conformational change of serum albumin occurring at pH... reversibility of the HSA structure after acid denaturation 1.5 Formation of hollow fiber membranes by phase inversion Compared to the flat sheet membrane configuration, the hollow fiber configuration offers several advantages, namely a high membrane area per unit volume of membrane module, self-support ability, flexibility and ease of handling during module preparation and operation [51] Therefore, the... that the degradation in the separation factor was caused by dilution 11 of the D-tryptophan remaining in the feed Moreover, they also have demonstrated that the separation factor can be further increased to around 20.0 by employing the twostage affinity UF system 1.4.3 Selection of stereoselective ligand Generally, the criteria for selection of the stereoselective ligand in the affinity UF system are as