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

i

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.

ii

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

iii

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

iv

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.

v

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

vi

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) L-tryptophan 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

vii

List of Symbols

A membrane area (m2)

cf neutral solute concentration in feed (mol/m3)

Cf,D D-enantiomer concentration in feed (mol/m3)

Cf,HSA HSA concentration in feed (mol/m3)

Cf,L L-enantiomer concentration in feed (mol/m3)

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)

viii

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)

1

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

2

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].

3

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]

4

Table 1.1 Comparison of existing enantioseparation techniques [19]

No. Methods Advantages Disadvantages 1. 1.1 1.2

Crystallization Direct or preferential crystallization [8-9] Diastereomeric crystallization [10-11]

Simplicity Low cost Wide applicability

Batch operation Expensive

2. 2.1 2.2

Kinetic resolution Chemical-mediated [12-13] Enzyme-mediated [14-15]

High stability High efficiency

Low efficiency Degrading enzyme activity

3. 3.1 3.2

Chromatography Supercritical fluid chromatography [16] Simulated moving bed chromatography [17-18]

High efficiency Low cost Continuous operation

Low capacity

4. Membrane [19-36] Low cost, energy efficient, high capacity, continuous operation, easy scale-up

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 non-

enantioselective membranes [19-20].

5

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 diffusion-

selectivity 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.

6

1.3.2 Non-enantioselective membranes

Different from the enantioselective membranes, the separation mechanism for non-

enantioselective 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 non-

enantioselective membranes are illustrated in Fig 1.2.

Figure 1.2 Separation mechanisms of enantioselective and non-enantioselective membranes

7

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

8

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:

,

,

,

,

p D

p L

f D

f L

CCCC

α = (1.1)

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.

, ,

, ,

100p D p L

p D p L

C Cee

C C−

= ×+

(1.2)

9

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:

vm

JPPµ×

=∆

(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:

VJA 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 D-

tryptophan over L-tryptophan as high as 7 was attained, which demonstrates the

feasibility of an affinity UF system for chiral separation. Interestingly, when the

10

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-LBSA + L BSA-L K = (1.5)

BSA L

BSA-DBSA + D BSA-D K = (1.6)

BSA D

↔

↔

• Stereospecific complexation constant

11

[ ][ ][ ]

[ ] [ ] [ ] [ ][ ] [ ] [ ][ ] [ ] [ ] [ ]

0

0

0

S-LS + L S-L K = (1.7)

S L

BSA = BSA + BSA-L + BSA-D (1.8)

D = D + BSA-D (1.9)

L = L + BSA-L + S-L (1.10)

L↔

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 L-

tryptophan 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 L-

tryptophan 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

12

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 two-

stage 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

13

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 warfarin-

azapropazone 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 L-

tryptophan 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].

14

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.

15

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

16

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.

17

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

18

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

19

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

20

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

21

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 1.32 0.395logsr MW= − + (2.1)

22

The solute rejection (RT) can be expressed by a log-normal probability function of

solute size [68] as shown in the following equation:

2( /2)11 ( )2

yp u du

Tf

cR erf y e

c π−

−∞

= − = = ∫ (2.2)

ln lnlns s

g

ry µσ−

= (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]:

( ) ( )( )

2

2

ln ln1 expln 2 2 ln

T p p p

p p p p

dR r rdr r

µ

σ π σ

− = −

(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

23

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⁄Cp,L Cf,L⁄ (2.5)

YD = Cp,D

Cf,D X 100% (2.6)

YL = Cp,L

Cf,L X 100% (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

24

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].

25

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 = np,D nf,D⁄np,L nf,L⁄ (2.8)

YL,o = np,L

nf,L 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

26

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,HSACf,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

27

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 10 x CHSA x n x l

(2.11)

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]:

% helix = �MRE222−234030300

�X 100% (2.12)

28

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 Fiber-4.5cm Fiber-10cm Fiber-15cm

Dope composition 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) 0.8

Air gap (cm) 4.5 10 15

Take up rate (cm/min) 1240

External coagulant Water

Spinning temperature (°C) 25

Coagulation bath temperature (°C) 25

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].

29

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

(L/(m2 bar h))

Mean pore radius

(nm) Standard deviation MWCO

(kDa) 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

30

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.

31

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.

D-tryptophan

L-tryptophan

32

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.

33

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

factor (αo)

Overall L-tryptophan yield

(YL,o)

Percentage of recovered

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 HSA-

tryptophan mixture solution is enhanced by the continuous addition of phosphoric

acid and sodium hydroxide.

34

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, acid-

denatured and base-renatured HSA samples are summarized in Table 3.4.

35

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

(deg cm2/dmol) % helix

MRE at 262 nm

(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 ion-

protein 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 α-

helical content in Table 3.4 shows a declining trend with increasing denaturation-

renaturation 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

36

medium pH is readjusted back to pH 9.0. However, the ellipticity value of the base-

renatured 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 denaturation-

renaturation 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

37

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.

38

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 denaturation-

renaturation 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

39

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 denaturation-

renaturation experiment.

40

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.

41

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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