PROTEIN TRANSPORT AND FOULING BEHAVIOR OF …
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The Pennsylvania State University The Graduate School College of Engineering PROTEIN TRANSPORT AND FOULING BEHAVIOR OF ZWITTERIONIC ULTRAFILTRATION MEMBRANES A Thesis in Chemical Engineering by Mahsa Hadidi © 2014 Mahsa Hadidi Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science May 2014
Transcript of PROTEIN TRANSPORT AND FOULING BEHAVIOR OF …
The Pennsylvania State University
The Graduate School
College of Engineering
PROTEIN TRANSPORT AND FOULING BEHAVIOR OF ZWITTERIONIC
ULTRAFILTRATION MEMBRANES
A Thesis in
Chemical Engineering
by
Mahsa Hadidi
© 2014 Mahsa Hadidi
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science
May 2014
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The thesis of Mahsa Hadidi was reviewed and approved* by the following:
Andrew L. Zydney
Head of the Department of Chemical Engineering
Walter L. Robb Chair and Professor of Chemical Engineering
Thesis Advisor
Ali Borhan
Professor of Chemical Engineering
Themis Matsoukas
Professor of Chemical Engineering
*Signatures are on file in the Graduate School.
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ABSTRACT
The need for robust, cost-effective, and high-resolution methods for the
purification of recombinant proteins has created a need for ultrafiltration membranes with
lower fouling behavior and high selectivity. Several recent studies have shown that
zwitterionic membranes can be low fouling while retaining some of the benefits of
electrically-charged membranes in terms of their high permeability and selectivity. The
overall objective of this thesis was to develop a more fundamental understanding of the
performance characteristics of zwitterionic membranes by comparing the properties of a
series of charged, neutral, and zwitterionic membranes with similar pore size /
permeability.
The surface-modified membranes were generated by covalent attachment of small
ligands to a base cellulose membrane. The membranes were characterized using
streaming potential measurements, XPS, protein fouling, and protein transmission. The
latter was examined using lysozyme and α-lactalbumin charge ladders, which consist of a
series of chemical derivatives of the base protein differing by single charge groups that can
be analyzed by capillary electrophoresis. The sieving coefficients were analyzed using
available hydrodynamic models based on the partitioning of a charged sphere in a
charged cylindrical pore.
The zwitterionic membranes showed minimal protein adsorption and a very low
degree of protein fouling over a wide range of conditions. The fouling behavior of the
charged membranes was strongly influenced by electrostatic interactions, although the
zwitterionic membrane had a lower degree of fouling than the charged membranes even
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when the protein and membrane had like polarity. The low protein fouling characteristics,
coupled with their high selectivity, makes these zwitterionic membranes promising
candidates for high performance ultrafiltration processes.
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Table of Contents
LIST OF FIGURES ....................................................................................................... ix
LIST OF TABLES ....................................................................................................... xii
Chapter 1 .........................................................................................................................1
1.1 Background .......................................................................................................1
1.2 Membrane technology for protein purification ...................................................2
1.3 Thesis Program ..................................................................................................6
Chapter 2 .........................................................................................................................8
2.1 Introduction .......................................................................................................8
2.2 Bulk Mass Transport .........................................................................................9
2.2.1 Concentration Polarization - Stagnant Film Model......................................9
2.2.2 Bulk Mass Transfer Coefficient ................................................................ 12
2.3 Membrane Transport ....................................................................................... 13
2.3.1 Solvent Transport - Membrane Hydraulic Permeability ............................ 13
2.3.2 Solute Transport - Thermodynamic Contributions .................................... 14
2.4 Protein Net Charge Analysis ............................................................................ 19
2.4.1 Protein Charge Calculations from Amino Acid Composition .................... 19
2.4.2 Protein Charge from Capillary Electrophoresis-Electrophoretic Mobility .. 21
Chapter 3 ....................................................................................................................... 23
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3.1 Introduction ..................................................................................................... 23
3.2 Membranes ...................................................................................................... 23
3.2.1 Membrane Materials ................................................................................. 23
3.2.2 Membrane Modification ........................................................................... 25
3.2.3 Streaming potential measurements ............................................................ 28
3.3 Solution Preparation ........................................................................................ 31
3.3.1 Buffer solutions ........................................................................................ 31
3.3.2 Protein Solutions ...................................................................................... 32
3.3.3 Dextran solutions...................................................................................... 34
3.4 Ultrafiltration .................................................................................................. 35
3.4.1 Apparatus ................................................................................................. 35
3.4.2 Membrane Hydraulic permeability ........................................................... 36
3.4.3 Protein Sieving ......................................................................................... 37
3.4.4 Diafiltration .............................................................................................. 38
3.4.5 Protein Fouling ......................................................................................... 38
3.5 Size Exclusion Chromatography (SEC) ........................................................... 39
3.6 Capillary electrophoresis ................................................................................. 40
3.7 X-ray Photoelectron Spectroscopy (XPS) ........................................................ 41
Chapter 4 ....................................................................................................................... 42
4.1 Introduction ..................................................................................................... 42
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4.2 Materials and Methods .................................................................................... 42
4.3 Results and Discussions ................................................................................... 43
4.3.1 Membrane Modification ........................................................................... 43
4.3.2 Membrane Surface Charge Characteristics................................................ 46
4.3.3 Dextran Ultrafiltration .............................................................................. 50
4.3.4 Static adsorption ....................................................................................... 51
4.3.5 IgG ultrafiltration ..................................................................................... 54
4.3.6 Protein ultrafiltration ................................................................................ 62
4.4 Conclusions ..................................................................................................... 64
Chapter 5 ....................................................................................................................... 66
5.1 Introduction ..................................................................................................... 66
5.2 Materials and Methods .................................................................................... 67
5.3 Results and Discussions ................................................................................... 67
5.3.1 Membrane Characterization ...................................................................... 67
5.3.2 Charge ladder Characterization ................................................................. 68
5.3.3 Ultrafiltration Experiments ....................................................................... 73
5.4 Conclusions ..................................................................................................... 77
6.1 Introduction ..................................................................................................... 80
6.2 Protein Transport through Surface Modified Membranes ................................. 81
6.3 Protein Fouling of Surface Modified Membranes............................................. 82
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6.4 Recommendations ........................................................................................... 83
References ..................................................................................................................... 85
Appendix ....................................................................................................................... 93
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LIST OF FIGURES
Figure 2.1 Schematic of concentration polarization during protein ultrafiltration. ...... 10
Figure 3.1 Molecular structure of cellulose. .............................................................. 24
Figure 3.2 Scanning electron micrograph showing the cross section of the composite
regenerated cellulose membrane. Taken from Burns (2000) with
permission. .............................................................................................. 25
Figure 3.3 Schematic of the reaction chemistry used to generate the surface modified
cellulose membranes (second reaction shown with the zwitterionic ligand).
................................................................................................................ 27
Figure 3.4 Molecular structure of the chemically-modified membranes where R is the
glucose monomer in the base cellulose. .................................................... 28
Figure 3.5 Schematic of the streaming potential apparatus used to determine the
effective membrane surface charge. ......................................................... 30
Figure 3.6 Schematic representation of the acylation reaction using acetic anhydride
(reproduced with permission from Ebersold and Zydney, 2004). .............. 34
Figure 3.7 Schematic of experimental set-up for constant pressure ultrafiltration
experiments.............................................................................................. 36
Figure 4.1 XPS spectra showing the nitrogen peak for all 100 kDa modified
membranes. .............................................................................................. 44
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Figure 4.2 XPS spectra showing the sulfur peak for all 100 kDa modified membranes.
................................................................................................................ 44
Figure 4.3 Streaming potential data for 100 kDa Ultracel™ zwitterionic membrane in
10 mM buffered KCl solutions at several pH. ........................................... 48
Figure 4.4 Correlation between the apparent zeta potential and the calculated charge
based on the pKa values of the lysine ligand. ............................................ 49
Figure 4.5 Dextran sieving coefficients in 150 mM ionic strength at pH 7 using
different surface-modified membranes. .................................................... 51
Figure 4.6 Amount of protein adsorption from 5 g/L IgG at pH 9 on different modified
membranes. .............................................................................................. 52
Figure 4.7 Filtrate flux as a function of time during a typical fouling experiment for
the zwitterionic membrane using a 5 g/L IgG solution in 10 mM buffered
KCl at pH 7 and a constant pressure of 69 kPa (10 psi). ........................... 55
Figure 4.8 Filtrate flux (top panel) and filtrate concentration (bottom panel) for
ultrafiltration of a 5 g/L IgG solution in 10 mM buffered KCl at pH 5 at a
constant pressure of 69 kPa through the zwitterionic, positive, and negative
(sulfonic acid) membranes. ...................................................................... 59
Figure 5.1 Capillary electropherograms for α-lactalbumin (top panel) and lysozyme
(bottom panel) charge ladders in 10 mM tris/glycine buffer at pH 8.3. ..... 70
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Figure 5.2 Net charge for the first seven peaks in the α-lactalbumin and lysozyme
charge ladders evaluated both from the electrophoretic mobility data using
10 mM tris/glycine buffer at pH 8.3 (open symbols) and from the amino
acid composition (filled symbols)............................................................. 73
Figure 5.3 Observed sieving coefficients for ultrafiltration of lysozyme charge ladder
at pH 7 through 100 kDa modified Ultracel™ membrane as a function of
net protein charge. .................................................................................... 75
Figure 5.4 Observed sieving coefficients as a function of charge interaction parameter
(product of the dimensionless surface charge densities for the protein and
the membrane) for different membranes. Filled symbols represent the
experimental data with the solid curves representing the theoretical model.
................................................................................................................ 77
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LIST OF TABLES
Table 2.1 Expansion coefficients for Kt and Ks functions in Equation 2.9 and 2.10.16
Table 3.1 Physicochemical properties of proteins. ..................................................... 33
Table 4.1 Atomic composition (percent) and calculated degree of modification for
different 100 kDa modified membranes determined from XPS data. .......... 46
Table 4.2 The experimental values for the apparent zeta potential of surface-modified
100 kDa Ultracel™ membranes at pH 7 in 10 mM buffered KCl. .............. 50
Table 4.3 Effect of protein adsorption on the permeability of the surface-modified 100
kDa Ultracel™ membranes........................................................................ 54
Table 4.4 Buffer flux (after protein adsorption) and initial filtrate flux with a 5 g/L IgG
solution for the different membranes at pH 5, 7, and 9. .............................. 57
Table 4.5 Flux recovery of different modified 100 kDa membranes after 1 hr
ultrafiltration of an IgG solution at pH 5, 7, and 9. ..................................... 61
Table 4.6 Initial buffer flux and flux recovery ratio for the zwitterionic and positively-
charged membranes after ultrafiltration of a 5 g/L IgG solution at pH 5 with
and without a pre-adsorption step. ............................................................. 62
Table 4.7 Flux recovery of zwitterionic and positively-charged 100 kDa membranes
after 1 hr ultrafiltration of lysozyme and α-lactalbumin at pH 7 and BSA at
pH 4.7. ...................................................................................................... 63
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Table 5.1 Apparent zeta potential of surface-modified 100 kDa Ultracel™ membranes
at pH 7 in 10 mM buffered KCl. ................................................................ 68
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Chapter 1
Introduction
1.1 Background
The production of high value recombinant proteins requires robust, cost-effective,
and high-resolution purification methods that can provide high yield and purification of
the desired product. Although there can be considerable variability in the economics for
different therapeutic proteins, several studies have reported that up to 80% of the total
manufacturing cost is associated with the downstream purification process (Clark and
Blanch, 1997). This is most pronounced in the production of high-dose therapeutic
proteins, e.g., monoclonal antibodies used in the treatment of various forms of cancer and
a number of immunologic disorders (van Reis and Zydney, 2007; Zydney, 2009). Current
annual demand for some antibody products is on the order of 1000 kg, creating a number
of new separation challenges for the downstream purification process (Thömmes and
Etzel, 2007; Zydney, 2009). There is tremendous interest in the development of improved
separation technologies to meet the needs of the evolving biotechnology industry.
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1.2 Membrane technology for protein purification
Membranes are very attractive for the separation and purification of high value
proteins since they operate under mild conditions that will not degrade or damage the
biological products. In addition, membranes have robust performance, high throughput,
easy scale-up, low energy consumption, low space requirements, and environmentally
friendly operating characteristics (no need for toxic chemicals) (Guo et al., 2012; Li et al.,
2012a). Ultrafiltration (UF) membranes with pore size from around 1 to 15 nm are widely
used for protein concentration and buffer exchange; UF is the method of choice for final
formulation of nearly all recombinant protein products (van Reis and Zydney, 2007).
UF was originally viewed as a purely size-based separation process with the
proteins retained by the membrane due to steric exclusion from the small pores.
However, as early as 1975, Chang et al. (Chang et al., 1975) reported that the
transmission of a synthetic polyanion (dextran sulfate) through the renal glomerular
capillary was about 20-fold smaller than that of a neutral dextran of similar size and
structure. Malone and Anderson (Malone and Anderson, 1978) attributed the observed
reduction in the hindered diffusion coefficient of latex particles through track-etched
mica membranes at low salt concentration to strong electrostatic interactions at low ionic
strength. Subsequent studies have extended these observations to pressure-driven
filtration of charged proteins through a range of commercial ultrafiltration membranes
with different surface charge characteristics and under different solution conditions. For
example, Mehta and Zydney (Mehta and Zydney, 2006) showed that the transmission of
a positively-charged protein through a series of positively charged membranes was a
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strong function of membrane charge. Pujar and Zydney (Pujar and Zydney, 1994)
reported that the transmission of negatively charged bovine serum albumin through a
negatively charged membrane decreased by more than two orders of magnitude at low
ionic strength due to the absence of significant electrostatic shielding under these
conditions. These results clearly demonstrate that protein transport through
semipermeable ultrafiltration membranes is strongly affected by electrostatic interactions
between the charged membrane and the charged protein.
Electrostatic effects have also been exploited to achieve high resolution protein
separations, with the charged membranes providing high retention of like-charged
proteins while allowing relatively uncharged solutes to pass into the permeate (van Reis
and Zydney, 2007). Examples include the separation of bovine serum albumin (BSA)
from hemoglobin (van Eijndhoven et al., 1995), the separation of BSA and
immunoglobulin G (Saksena and Zydney, 1994), the purification of an antigen binding
fragment from BSA (Van Reis et al., 1999), the purification of a monoclonal antibody
from Chinese Hamster Ovary host cell proteins (van Reis and Zydney, 2007), and the
purification of an antibody fragment from E. Coli host cell proteins (Lebreton et al., 2008).
Electrostatic interactions can also be used to develop high performance ultrafiltration
membranes with significantly greater protein retention (or selectivity) for a given value of
the permeability than conventional UF membranes (Mehta and Zydney, 2006; Zydney,
2009).
One of the challenges in many UF processes is membrane fouling due to specific
interactions between the proteins and membrane surface. Fouling causes a decline in the
filtrate flux, increasing the required feed pressure and requiring additional membrane
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cleaning (Guo et al., 2012). A variety of strategies are used to control / minimize fouling
including modification of the membrane surface to reduce protein – membrane interactions
(Pasmore et al., 2001; Vrijenhoek et al., 2001). Several recent studies have focused on the
development of non-porous zwitterionic surfaces, analogous to the zwitterionic character
(presence of both positive and negative charge groups in close proximity) of phospholipid
cell membranes (Chen et al., 2005; Ishihara and Takai, 2009; Lewis, 2000; Vermette and
Meagher, 2003). The low fouling behavior of these zwitterionic materials is attributed to
their ability to bind a significant number of water molecules through both electrostatic
interactions and hydrogen bonding (Chen et al., 2005; Harder et al., 1998; Ishihara et al.,
1998; Li et al., 2012b; Nagumo et al., 2012; Sun et al., 2006). This hydration layer
effectively excludes the protein from the surface thus preventing adsorption (He et al., 2008;
Ishihara et al., 1998).
The low fouling characteristic of zwitterionic surfaces has also been exploited in the
development of porous membranes. For example, Jiang and co-workers showed that
membranes made from a zwitterionic sulfobetaine copolymer with either polyethersulfone
(Wang et al., 2006) or acrylonitrile (Sun et al., 2006) were highly hydrophilic and more
resistant to protein fouling than the base membranes during ultrafiltration of bovine serum
albumin (BSA). Li et al. (Li et al., 2012b) obtained similar results by grafting the
zwitterionic monomer sulfobetaine methacrylate onto the surface of poly(vinylidene
fluoride) (PVDF) membranes. An et al. (An et al., 2013) used zwitterionic amine monomers
to make thin-film composite nanofiltration membranes. The relative flux recovery ratio after
BSA ultrafiltration increased from 0.83 to 0.93 by increasing the zwitterionic monomer
concentration from 0 to 3.2 mol%. Ji et al. (Ji et al., 2012) introduced a novel zwitterionic
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terpolymer to nanofiltration membranes, significant increasing the water flux recovery after
BSA ultrafiltration. Zwitterionic grafted PVDF hollow fiber membranes show high
hydrophilicity and strong resistance to both protein and oil fouling (Li et al., 2012c).
Zwitterionic grafted PES hollow fiber membranes have shown improved antifouling
behavior in comparison with the un-modified membrane for both BSA and lysozyme (Razi
et al., 2012).
In addition to the use of long-chain zwitterionic polymers, several studies have
demonstrated the potential of using small zwitterionic ligands, e.g., peptides and amino
acids, for membrane surface modification (Chelmowski et al., 2008; Shi et al., 2011). For
example, Shi et al. (Shi et al., 2011) grafted the amino acids lysine, glycine, and serine onto
the surface of a hydrolyzed polyacrylonitrile membrane containing a high concentration of
carboxylic acid groups. All of the modified membranes had similar hydrophilicity, as
determined from water contact angle measurements, but the lysine-modified membranes
showed the least protein fouling under both static and dynamic (filtration) conditions.
However, it is difficult to interpret these results since all of the membranes had a significant
negative charge due to the presence of the carboxylic acids in the hydrolyzed
polyacrylonitrile membrane.
Although these recent studies have clearly demonstrated the potential of zwitterionic
ultrafiltration membranes, there are still considerable uncertainties regarding the factors
controlling the performance of these membranes and how they compare to more
conventional charged and neutral membranes. Most of the early studies in this area were
performed using zwitterionic polymers, in which case the modified membranes tended to
have very different permeability, pore size, and even surface morphology than the
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unmodified membranes. The work by Shi et al. (Shi et al., 2011) used small ligands to
eliminate many of these effects, but the base membrane had a significant negative charge
which persisted in the modified membranes. In addition, none of these studies examined
protein transmission through the zwitterionic membranes, making it impossible to
determine whether these low fouling membranes had the combination of selectivity and
permeability that is needed for high performance ultrafiltration processes.
1.3 Thesis Program
The overall objective of this thesis was to develop a more fundamental
understanding of the performance characteristics of zwitterionic membranes by comparing
the properties of these membranes with a series of charged and neutral membranes with
similar pore size / permeability. This included: (1) studying the fouling characteristics of
these membranes during both static protein adsorption and actual ultrafiltration over a
range of solution pH, and (2) investigating the effects of electrostatic interactions on
protein transport through these membranes using protein charge ladders, which consist of
a series of chemical derivatives of a given protein differing only by single charge groups.
The general theoretical background used to analyze the ultrafiltration results is
presented in Chapter 2. This includes a brief review of available theoretical analyses for
solute and solvent transport through membranes with relatively small pores, with a
particular emphasis on the effects of electrostatic interactions on solute partitioning into
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charged membrane pores. The last part reviews the calculation of the protein net charge
from both the electrophoretic mobility and the known amino acid sequence.
Chapter 3 describes the experimental set-up, materials, and methods used in the
experimental studies described in this thesis. Specific details on some of the
experimental procedures are provided in the appropriate Chapters.
Chapter 4 presents membrane fouling studies under both static exposure and
dynamic ultrafiltration using human serum IgG as a model protein. Limited data are also
provided with BSA, α-lactalbumin, and lysozyme.
Chapter 5 examines the transport of protein charge ladders through zwitterionic,
positively-charged, acidic, and hydrophilic membranes generated by covalent
modification of a base cellulose membrane using specific small ligands. Ultrafiltration
data were analyzed using available theoretical models describing the partitioning of a
charged sphere in a charged cylindrical pore.
Chapter 6 summarizes the major contributions of this thesis and makes several
recommendations for future studies on the development of high performance zwitterionic
ultrafiltration membranes for protein purification.
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Chapter 2
Theoretical Background
2.1 Introduction
This Chapter provides a brief review of the theoretical models developed to
describe the basic mass transport and separation phenomena governing the behavior of
ultrafiltration systems with an emphasis on electrostatic interactions. Previous reviews of
this theoretical analysis have been presented by Zeman and Zydney (Zeman and Zydney,
1996) and in several dissertations published under the direction of Professor Andrew
Zydney (Burns, 2000; Mehta, 2006; Molek, 2008). The discussion below draws
extensively from these prior reviews.
The overall rate of protein transport through a semipermeable ultrafiltration
membrane is determined by the rate of protein transport from the bulk solution to the
membrane and through the membrane pores. Transport in the bulk solution is governed
primarily by the system hydrodynamics while transport through the membrane pores has
contributions from both thermodynamics (including electrostatic interactions) and
hydrodynamics.
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2.2 Bulk Mass Transport
2.2.1 Concentration Polarization - Stagnant Film Model
The pressure-driven flow through a semipermeable membrane causes an
accumulation of the completely or partially retained solute at the upstream surface of the
membrane. This phenomenon is called concentration polarization and causes the protein
(solute) concentration to vary from the value in the bulk solution ( ) to a much greater
value at the membrane surface/wall ( over the distance of the concentration
polarization boundary layer thickness, . Figure 2.1 shows a schematic of the
concentration polarization phenomenon, including the expected concentration profile.
The high concentration at the upstream surface of the membrane can significantly affect
the overall system behavior. It increases the driving force for protein transport into and
through the membrane, while reducing the effective pressure driving force for solvent
transport due to the osmotic pressure associated with the retained protein. The high
protein concentration at the membrane surface can also contribute to an increase in
membrane fouling.
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Figure 2.1 Schematic of concentration polarization during protein ultrafiltration.
Concentration polarization in membrane systems is most commonly analyzed
using the stagnant film model. This model approximates the concentration profile
upstream of the membrane by treating the problem as a one-dimensional (stagnant)
boundary layer, neglecting the complexities associated with the detailed fluid flow
characteristics in the particular module as well as the coupling between mass and
momentum transport. In the classical model, protein-protein interactions are neglected
and the solute diffusivity and viscosity are both assumed to be independent of the solute
concentration and constant throughout the boundary layer. At steady state, the net solute
flux towards the membrane is set equal to the solute flux through the membrane and into
the filtrate solution:
[2.1]
𝐶𝑏
𝐶𝑤
𝐶𝑓
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where is the filtrate flux through the membrane (equal to the volumetric filtrate flow
rate per unit membrane area), is the concentration of solute in the filtrate solution, is
the local solute concentration at a position above the membrane surface, and is the
free solution diffusion coefficient of the solute. Equation 2.1 is integrated across the
concentration boundary layer (from at to at ) giving the
following expression for the filtrate flux:
(
) [2.2]
A more detailed analysis of this model is provided by Zydney (Zydney, 1997).
Equation 2.2 can also be used to evaluate the effect of the filtrate flux on the
observed sieving coefficient (
. The observed sieving coefficient is typically
evaluated in terms of the actual sieving coefficient, which is defined as ratio of the solute
concentration in the filtrate to that at the membrane wall ( as:
(
)
(
) [2.3]
At low filtrate flux (
), the observed sieving coefficient is equal to the actual sieving
coefficient since concentration polarization is minimal. increases with increasing filtrate
flux, approaching a value of one as .
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2.2.2 Bulk Mass Transfer Coefficient
The solute mass transfer coefficient, , is defined as the ratio of the solution
diffusion coefficient ( ) to the boundary layer thickness ( ) in Equations 2.2 and 2.3.
This coefficient is a function of the solute diffusivity and the hydrodynamics of the
particular membrane device. Although it is possible to evaluate the mass transfer
coefficient theoretically for relatively simple module configurations, the analysis of mass
transfer in the stirred cells used in this research is difficult due to the complex flow
profiles in this system. Instead, the mass transfer coefficient is typically evaluated using a
semi-empirical equation for mass transfer in a stirred cell for laminar flow (
) that was developed by Smith et al. (Smith et al., 1968) based on the rate of
benzoic acid dissolution into a stirred solution as:
[2.4]
where
is the Sherwood number,
is the Reynolds number,
is
the Schmidt number, is the radius of the stirred cell, is the stirring speed, and is the
kinematic viscosity. Opong and Zydney (Opong and Zydney, 1991) evaluated as 0.23
for the 25 mm diameter Amicon stirred cell used in this thesis based on data for the filtrate
flux as a function of the transmembrane pressure at several bulk protein concentrations and
stirring speeds. The protein diffusion coefficient ( in m2/s) can be evaluated as (Young
et al., 1980):
[2.5]
13
where is the solution viscosity (in Pa s), is the absolute temperature (in K), is the
Boltzmann’s constant , and is the protein molecular weight in
g/mol. Equation 2.5 is valid only at infinite dilution because it neglects the effects of
protein-protein interactions.
2.3 Membrane Transport
The rate of solute and solvent transport through porous membranes is typically
described using hydrodynamic theories in which the membranes are modeled as an array
of well-defined cylindrical pores, while the solutes are considered to behave as uniform
rigid spheres (Anderson and Quinn, 1974; Deen, 1987). The advantage of the
hydrodynamic models is that the key transport parameters can be calculated directly in
terms of the physical properties of the solute and the pores. Hydrodynamic theories can
be easily extended to incorporate the effects of a pore size distribution (by numerical
integration over the distribution) as well the effects of electrostatic interactions
(Mochizuki and Zydney, 1993; Saksena and Zydney, 1995).
2.3.1 Solvent Transport - Membrane Hydraulic Permeability
The rate of solvent transport through a membrane is generally described in terms
of the hydraulic permeability ( ):
14
[2.6]
where is the transmembrane pressure.
The rate of solvent transport is also dependent on the membrane surface charge
and solution ionic strength due to electrokinetic effects. The presence of a net surface
charge on the pore wall causes an accumulation of counter-ions in the electrical double
layer adjacent to the pore wall. The pressure-driven convective fluid flow through the
charged pore will generate an unequal flux of the co-ions and counter-ions, leading to the
development of an induced (streaming) potential. At steady state, the streaming potential
generates a back conductive ion transport that exactly balances the convective ion flux,
resulting in a situation in which there is no net current flow through the pore. The
induced streaming potential reduces the magnitude of the solvent flux due to the net force
on the fluid exerted by the action of the electric field on the ions (often referred to as
counter-electroosmosis). A detailed review of solvent transport through electrically-charged
membranes is provided elsewhere (Burns, 2000; Pujar and Zydney, 1994).
2.3.2 Solute Transport - Thermodynamic Contributions
The rate of protein transport through small pore ultrafiltration membranes is
typically analyzed in terms of both thermodynamic and hydrodynamic interactions, with
the actual protein sieving coefficient ( ) expressed as:
[2.7]
15
where is the thermodynamic equilibrium partition coefficient between the bulk solution
and the membrane pore and is the hindrance factor for convection, which accounts for the
additional hydrodynamic drag on the solute molecule due to the presence of the pore wall.
Equation 2.7 assumes that protein transport is dominated by convection, which is a
reasonable approximation during protein ultrafiltration due to the relatively high Peclet
numbers in these systems.
The hindrance factor for convection, , can be evaluated in terms of an integral
over the radial coordinate in the pore. For a solute located at the pore axis (i.e., at the pore
centerline), the integral becomes (Deen, 1987):
[2.8]
where is the ratio of the solute to pore radius. Expressions for Ks and Kt can be
developed using matched asymptotic expansions (Bungay and Brenner, 1973) giving:
√
[ ∑ ] ∑
[2.9]
√
[ ∑ ] ∑
[2.10]
with the expansion coefficients provided in Table 2.1.
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Table 2.1 Expansion coefficients for Kt and Ks functions in Equation 2.9 and 2.10.
Subscript, n
1 -73/60 7/60
2 77,293/50,400 -2,227/50,400
3 -22.5083 4.0180
4 -5.6117 -3.9788
5 -0.3363 -1.9215
6 -1.216 4.392
7 1.647 5.006
The equilibrium partition coefficient is defined as the ratio of the average protein
concentration in the pore to that in the bulk external solution immediately adjacent to the
membrane:
∫ [
]
[2.11]
where is the pore radius, is the radial coordinate within the cylindrical pore, is the
Boltzmann constant, is the absolute temperate, and is the total interaction
potential. Smith and Deen (Smith and Deen, 1980) developed the first rigorous analytical
expressions for the electrostatic potential for a spherical solute in a cylindrical pore by
solving the linearized Poisson-Boltzmann equation using matched asymptotic expansions
17
in cylindrical and spherical coordinates. The results for interactions at constant surface
charge density are conveniently expressed as:
[2.12]
The coefficients , , , and are all positive functions of the solution ionic
strength, solute radius, and pore radius:
[2.13]
[2.14]
[2.15]
[2.16]
[2.17]
∫ [(
) ]
[
]
[2.18]
where and are modified Bessel functions, is the dimensionless pore radius,
and is the inverse Debye length:
[ ∑
]
[2.19]
where and are the valence and concentration of each ion.
18
The dimensionless surface charge densities of the solute, , and pore, , are
defined as:
[2.20]
[2.21]
with the permittivity of free space , the dielectric constant of
the solution, the Faraday constant , and the ideal gas constant
. The dimensional surface charge densities of the solute and pore (
and ) can be evaluated in terms of the net electronic charge on the solute (Z) and the
apparent zeta potential of the membrane pore ( ), respectively, as:
[2.22]
(
) [2.23]
where is the electron charge and is the bulk electrolyte
concentration.
Equations 2.13 to 2.18 are valid for a solute located at the pore axis.
Corresponding results are available for arbitrary radial positions (Smith and Deen, 1983)
as well as for interactions at constant surface potential instead of constant surface charge
density (Smith and Deen, 1980, 1983). It is also possible to extend this analysis to
account for the effects of charge regulation using a linearized form of the charge
regulation boundary condition; the resulting equations account for the change in surface
19
charge / potential of the protein and pore wall associated with the alteration in the local
electrical potential field (and ion concentrations) when the protein enters the pore (Pujar
and Zydney, 1997).
2.4 Protein Net Charge Analysis
2.4.1 Protein Charge Calculations from Amino Acid Composition
The net electrical charge on a protein is determined by its amino acid sequence
due to the dissociation of the various ionizable residues on the surface of the protein
along with the adsorption (or binding) of specific ions from the bulk electrolyte. The
number of dissociated acidic and basic amino acid residues can be calculated from the
intrinsic dissociation constant for each amino acid. For example, the dissociation
equilibrium for an α-carboxylic acid can be expressed as:
[ ][ ]
[ ] [2.24]
Equation 2.24 can be rewritten in terms of the pH and the number of dissociated groups
( ) as:
[2.25]
where [ ], [
], and is the total number of titratable
species. The concentration at the protein surface (required in Equations 2.24 and 2.25)
is different from the bulk concentration due to electrostatic interactions between the
20
charged protein and the charged hydrogen ion. The local concentration at the membrane
surface can be related to the bulk concentration using a classical Boltzmann distribution:
[2.26]
where is the bulk hydrogen ion concentration, is the electron charge, and is the
electrostatic potential at the protein surface:
[2.27]
where is the net protein surface charge (evaluated as the number of electronic charges).
Equation 2.27 is developed assuming that the protein is a hard sphere with the electrical
charges distributed uniformly over the spherical surface (Overbeek and Wiersema, 1967).
The protein charge is equal to the difference between the maximum number of positive
charges (N-terminal, histidine, lysine, and arginine) and the sum of all the dissociated
groups:
∑
[2.28]
Equations 2.26 to 2.28 can be solved iteratively to evaluate the net protein charge
as a function of the bulk pH and solution ionic strength (which determines the Debye
length). The development of these equations is discussed in more detail by Menon and
Zydney (Menon and Zydney, 2000). The number and values of the various amino
acids present in the model proteins used in this thesis are summarized in Appendix.
21
2.4.2 Protein Charge from Capillary Electrophoresis-Electrophoretic
Mobility
The electrophoretic mobility reflects the balance between the electrical forces
arising from the applied electric field and the hydrodynamic (friction) forces associated
with the viscosity of the suspending medium. A large number of theoretical models have
been developed for the electrophoretic mobility, with the differences primarily in the
approximations made in evaluating the electrical interactions. These approximations
include both the detailed structure of the equilibrium electrical potential (e.g., the use of
the low electrical potential, small Debye length, or flat plate approximations) and the
evaluation of the distortion of the equilibrium structure associated with the particle and
fluid motion during electrophoresis.
A simple analytical expression for the electrophoretic mobility in terms of the
electrical potential at the surface of a hard sphere for the limiting case when the electrical
double layer thickness is much smaller than the particle radius ( ) and the electrical
potential is relatively small (Debye-Huckel approximation) is given as (Overbeek and
Wiersema, 1967):
[2.29]
where is the electrostatic potential at the particle surface, is the electrical permittivity
of the solution, and is the solution viscosity.
22
If the electrical double layer is much larger than the particle radius ( ), the
electrophoretic mobility is given by the Debye-Huckel equation assuming that the
potential is low:
[2.30]
Henry (Henry, 1931) obtained a more complete solution for the electrophoretic mobility
that accounts for the distortion of the electric field lines by the presence of the particle,
with the resulting expression valid over the entire range of Debye lengths:
[2.31]
with
[
] ∫
[2.32]
where is Henry’s function, which accounts for finite double layer thickness, and is a
dummy variable over which the integration is performed. The zeta potential for a
uniformly charged hard sphere can be expressed in terms of the particle net charge using
Equation 2.27. The net electrical charge of the protein is thus given as:
[2.33]
More details on the evaluation of the electrophoretic mobility from capillary
electrophoresis experiments are provided elsewhere (Menon, 1999; Molek, 2008).
23
Chapter 3
Materials and Methods
3.1 Introduction
This chapter describes the materials, apparatus, and methods used for the
experimental studies performed in this thesis. Additional details on specific materials or
methods are provided in subsequent chapters as appropriate.
3.2 Membranes
3.2.1 Membrane Materials
Asymmetric membranes are used in almost all commercial applications of
ultrafiltration. These membranes are anisotropic and have a thin skin, which provides the
membrane its functionality, and a much thicker and more porous support that provides the
membranes its structural integrity. The small thickness of the skin allows much higher
fluxes to be obtained compared to symmetric membranes with comparable selectivity.
Although a variety of polymers can be used to make asymmetric ultrafiltration
membranes, regenerated cellulose is one of the most attractive membrane materials.
24
Cellulosic membranes have become a major component of the downstream purification
process for therapeutic proteins in the biotechnology industry. The free hydroxyl groups on
the glucose rings within the cellulose polymer renders the membrane highly hydrophilic,
significantly reducing protein binding and fouling during use. These hydroxyl groups are
also available for chemical modifications as discussed in the next section. Figure 3.1 shows
a schematic of the chemical structure of cellulose.
Figure 3.1 Molecular structure of cellulose.
Ultrafiltration experiments were performed using UltracelTM
composite
regenerated cellulose membranes with nominal molecular weight cut-off (MWCO) of
100 kDa provided by Millipore Corp. (Bedford, MA). UltracelTM
membranes with 10
kDa molecular weight cut-off were used for buffer exchange. The nominal molecular
weight cut-off refers to the molecular weight of a solute which has approximately 90%
rejection as determined by the manufacturer. A scanning electron micrograph (SEM) of
the cross section of the composite regenerated cellulose membrane is shown in Figure
3.2; the skin layer (approximately 0.5 - 1 μm thick) is not visible in the SEM. The two
layers seen in the SEM correspond to the porous cellulosic substructure (approximately
25
60 μm thick) and the porous polyethylene substrate on which the cellulose membrane is
cast.
Figure 3.2 Scanning electron micrograph showing the cross section of the composite
regenerated cellulose membrane. Taken from Burns (Burns, 2000) with
permission.
Membrane disks with 25 mm diameter were cut from large flat sheets using a
stainless-steel cutting device fabricated in our laboratory. All membranes were soaked in
90% (V/V) isopropanol for 45 min to remove any protective agents. The membranes
were then thoroughly rinsed with at least 100 L/m2 of deionized (DI) water.
3.2.2 Membrane Modification
Most of the approaches used for surface modification of cellulose membranes are
based on the activation and subsequent reaction of the free hydroxyl groups on the base
cellulose. Surface-modified ultrafiltration membranes were prepared from the Ultracel™
composite regenerated cellulose membranes by covalent attachment of different ligands
26
to epoxy-activated hydroxyl groups using the reaction chemistry developed by Liu et al.
(Liu et al., 2005) and subsequently modified by Mehta and Zydney (Mehta and Zydney,
2008) to avoid degradation of the cellulose (shown schematically in Figure 3.3). All
membranes were generated using the same linkage chemistry but with different ligands
having very similar chain length but with different end functionality. This made it
possible to study the effects of ligand chemistry on membrane surface characteristics
independent of the membrane pore size, providing an appropriate set of controls for
subsequent studies.
Membranes were first incubated in 0.1 M NaOH for 24 hr. The cellulose surface
was then activated by incubating the membrane in a 25 mL capped plastic jar for 2 hr at
45 oC in a mixture of epichlorohydrin (Alfa Aesar, A15823) and 0.1 M NaOH in a 1:2
ratio (by volume) (step 1 in Figure 3.3). The membranes were then carefully removed and
rinsed with DI water. The epoxide groups on the activated membranes were reacted with
the desired ligand (step 2 in Figure 3.3) by immersing in 20 mL of a 1 M solution of that
ligand: L-Lysine (Sigma, L5501) for the zwitterionic surface, hexamethylenediamine
(Sigma, H11696) for the positively-charged surface, 6-aminocaproic acid (Sigma,
A2504) for the acidic surface, 6-amino-1-hexanol (Sigma, A56353) for the hydrophilic
surface, and hexylamine (Sigma, 219703) for the hydrophobic surface. The molecular
structures of the different membranes are shown schematically in Figure 3.4 where R is
the glucose ring of the cellulose membrane. All reactions were conducted at 45°C for 12
hr to insure complete reaction with the epichlorohydrin groups. The membranes were
then removed from the reaction solution and thoroughly rinsed with DI water. Data were
also obtained with a negatively charged version of the Ultracel™ membrane generated by
27
direct chemical attachment of a ligand containing sulfonic acid groups to the free
hydroxyl of the cellulose (without activation with epichlorohydrin) as described
elsewhere (Van Reis, 2006).
Figure 3.3 Schematic of the reaction chemistry used to generate the surface modified
cellulose membranes (second reaction shown with the zwitterionic ligand).
28
Figure 3.4 Molecular structure of the chemically-modified membranes where R is the
glucose monomer in the base cellulose.
3.2.3 Streaming potential measurements
The effective surface charge of the different ultrafiltration membranes was
evaluated from streaming potential measurements using the approach described by Burns
and Zydney (Burns and Zydney, 2000). The membrane was placed between two
Plexiglass chambers (Figure 3.5), each filled with 10 mM buffered KCl solution at the
desired pH, taking care to remove all air bubbles. Ag/AgCl electrodes were then screwed
tightly into the ends of the chambers to ensure reproducible placement (approximately
29
0.1 – 0.2 cm away) relative to the membrane surface (with O-rings in place to provide
good seals).
The electrodes were prepared as follows. 1 mm diameter silver wires (Sigma
Chemical Co., St. Louis, MO) were lightly sanded and placed in a concentrated nitric
acid solution for approximately 10 s. Each wire was then washed with DI water and
placed in a 1 M KCl solution. A DC power supply was then connected to each silver
electrode and a steel wire, the current was set at 20 mA, and a uniform Ag/AgCl layer
was deposited on the wire surface for 20 min. Electrodes were stored in 0.5 M KCl
solution between experiments.
To measure the streaming potential of the membranes, an air-pressurized feed
reservoir containing an appropriate buffer solution was attached to the feed chamber. The
electrodes were connected to a Keithley 2000 Multimeter to evaluate the transmembrane
voltage as a function of the transmembrane pressure ( ), which was set by
pressurizing the feed chamber. The system was allowed to stabilize for approximately 15
to 30 min at each pressure before evaluating the voltage. The system pressure was
gradually increased from 14 to 34 kPa (2 to 5 psi), with data obtained at four or more
discrete pressures. The apparent zeta potential was evaluated from the slope of the
voltage (streaming potential) as a function of pressure using the Helmholtz-
Smoluchowski equation (Hunter, 1981):
[3.1]
where is the solution conductivity, is the permittivity of free space, is the dielectric
constant of the solution, and is the solution viscosity. Note that Equation 3.1 is only valid
30
under conditions where the double layer thickness is very small compared to the pore radius.
Thus, should be considered an apparent or effective zeta potential, which is directly
related to the membrane surface charge but may not be equal to the actual value. A more
detailed discussion on the evaluation of the membrane charge from the streaming potential
is provided by Burns (Burns, 2000).
Figure 3.5 Schematic of the streaming potential apparatus used to determine the
effective membrane surface charge.
31
3.3 Solution Preparation
3.3.1 Buffer solutions
Different buffer solutions were prepared by dissolving preweighed amounts of the
appropriate salts in deionized distilled water obtained from a NANOpure® Diamond
water purification system (Barnstead Thermolyne Corporation, Dubuque, IA) with a
conductivity less than 56 nS/cm. KCl powder (BDH Chemicals, BDH0258) and either 1
mM acetate (Sigma, S7670), Bis-Tris (MPBiomedicals, 101038), or borate (Sigma,
S9640) were used for pH 5, 7, and 9, respectively. The running buffer for the capillary
electrophoresis was prepared by adding 192 mM glycine (Sigma, G7403) and 25 mM
Trizma® base (Sigma, T1503) to a 10 mM KCl solution at pH 8.3. Higher ionic strength
solutions were prepared by dissolving appropriate amounts of KCl in tris/glycine
solution. All salts were analytical reagent grade. The solution pH was measured using a
Model 402 Thermo Orion pH meter (Beverly, MA) and adjusted using 0.1 M sodium
hydroxide (NaOH) or hydrochloric acid (HCl) as required. A 105 A plus conductivity
meter (Thermo Orion, Beverly, MA) was used to measure the solution conductivity. All
buffer solutions were filtered through a 0.2 μm pore size Supor® 200 membrane (Pall
Corporation, Ann Arbor, MI) prior to use to remove any particles or un-dissolved salt.
The ionic strength of the buffer solution was evaluated as:
∑
[3.2]
where and are the net charge and concentration of each ion, respectively.
32
3.3.2 Protein Solutions
Ultrafiltration experiments were performed with lysozyme and α-lactalbumin
charge ladders (described below). Fouling experiments were done using human serum
gamma globulin (IgG), bovine serum albumin (BSA), lysozyme, and α-lactalbumin as
model proteins. Table 3.1 summarizes the key physical properties and catalog numbers.
The protein isoelectric point and equivalent radius were taken from literature data. More
detailed information on the amino acid composition is provided in the Appendix.
Concentrated human serum IgG (obtained from SeraCare) was diluted with appropriate
buffer at the desired pH and ionic strength. Other protein solutions were prepared by
slowly dissolving pre-weighed amounts of protein powder in the desired buffer. The pH
of the protein solution was adjusted by adding appropriate amounts of 0.1 M acid or base
as required (e.g., HCl and KOH for buffered KCl solution). All protein solutions were
filtered through 0.22 μm Acrodisc® syringe filters (Pall Corporation) immediately prior
to use in the ultrafiltration experiments. Protein solutions were stored at 4°C and used
within 12 hr of preparation to minimize the likelihood of protein aggregation or
denaturation.
Protein concentrations were determined spectrophotometrically using a
SPECTRAmax Plus 384 UV-vis spectrophotometer (MD Corporation, Sunnyvale, CA)
with the absorbance evaluated at 280 nm for IgG and α-lactalbumin and 260 nm for
lysozyme. Actual concentrations were determined by comparison of the absorbance with
that of known protein standards.
33
Table 3.1 Physicochemical properties of proteins.
Protein
Molecular
weight,
(kDa)
Isoelectric
point
Equivalent
radius*, (nm)
Catalog
number
Lysozyme
(Rohani and Zydney, 2009)
14.3 11 1.6 Sigma
L6876
α-lactalbumin
(Molek and Zydney, 2007;
Rohani and Zydney, 2012)
14.2 4.6 1.6 Sigma
L5385
IgG
(Andersen et al., 2000;
Saksena and Zydney, 1994)
155 ≈7 5.5 Sera
Care
HS-475
BSA
(Menon and Zydney, 1998;
Razi et al., 2012)
67 4.7 3.45 Sigma
A7906
*Radius of sphere of equivalent volume
Protein charge ladders were synthesized by reaction of the lysine ɛ-amino groups
with acetic anhydride (Sigma, 242845) following the procedure described by Chung et al.
(Chung et al., 2009) as shown in Figure 3.6. The acetylated amide eliminates a potentially
protonable group by chemically blocking the lysine amino group. Thus, the resulting
charge ladder consists of a series of proteins with essentially the same size but each
differing by one or more charge groups. The lysozyme and α-lactalbumin charge ladders
were prepared by adding 1 M NaOH to a 10 g/L protein solution to bring the pH to 12.
Approximately 4 equivalents of 0.1 M acetic anhydride (per mole of protein) in 1,4-
dioxane (Sigma, 360481) were added to the solution, with the pH kept constant
throughout the 5 min reaction by addition of 0.1 N NaOH as needed. The pH was rapidly
34
lowered to 7 by addition of 1 M HCl to quench the reaction. The resulting protein
solution was diafiltered with at least four diavolumes of chilled DI water to remove the
dioxane, unreacted acetic anhydride, and any reaction by-products.
Figure 3.6 Schematic representation of the acylation reaction using acetic anhydride
(reproduced with permission from Ebersold and Zydney, (Ebersold and
Zydney, 2004)).
3.3.3 Dextran solutions
Dextrans are branched polymers made of glucose, joined by α-1,6 linkages in the
main chain and a small number of branches attached to the main chain by α-1,3 links.
They are synthesized naturally by a strain of the bacterium Leuconostoc mesenteroides.
Dextrans have been used extensively in the past for membrane characterization (Mehta
and Zydney, 2006; Mochizuki and Zydney, 1992) since they do not have any ionizable
side groups, thus providing a purely size-based measure of the membrane sieving
characteristics. The dextran diffusion coefficient is a function of the molecular weight as
evaluated by Granath (Granath, 1958):
[3.3]
where is the diffusion coefficient (in m2/s) and is the molecular weight in Da. The
Stokes radii can be evaluated from the diffusivity using the Stokes-Einstein equation to
give (Granath and Kvist, 1967):
35
[3.4]
with given in .
3.4 Ultrafiltration
3.4.1 Apparatus
Ultrafiltration experiments were performed in an Amicon 8010 stirred cell
(Millipore Corp., Bedford, MA). A membrane disc with effective area of 4.1 cm2 was
placed in the bottom of the stirred cell directly on top of a porous Tyvek® support that
provides membrane a mechanical support and minimizes deformation of the membrane at
high pressure. The stirred cell was placed on a magnetic stir plate with the stirring speed
set to 600 rpm using a Strobotac Type 1531-AB strobe light (General Radio Co.,
Concord, MA). An air-pressurized feed reservoir was connected to the stirred cell, with
the filtrate flux controlled by adjusting the pressure. A schematic of the apparatus is
shown in Figure 3.7.
36
Figure 3.7 Schematic of experimental set-up for constant pressure ultrafiltration
experiments.
3.4.2 Membrane Hydraulic permeability
The membrane hydraulic permeability ( ) was evaluated by measuring the
filtrate flux as a function of transmembrane pressure using a 10 mM buffered KCl
solution at pH 7. The permeability was evaluated from the slope of the data as:
[3.5]
where is the solution viscosity, is the filtrate flux, and is the transmembrane
pressure. Data were obtained at transmembrane pressures between 14 and 34 kPa (2 and
5 psi), with the filtrate flow rate evaluated by timed collection using a digital balance
(Model AG104, Mettler Toledo, Columbus, OH) with an accuracy of 100 μg.
37
3.4.3 Protein Sieving
Membrane sieving characteristics were evaluated using protein charge ladders to
directly study the effect of protein charge on the sieving coefficient. Each membrane was
first soaked in the charge ladder solution overnight at 4 °C to minimize initial transients
associated with protein adsorption on and within the membrane pores. The membrane
was then placed in the base of the stirred cell and the system was flushed with at least 25
L/m2 of buffer solution. The cell and feed reservoir were then filled with the charge
ladder solution, and the system was air-pressurized to approximately 10 kPa (1.5 psi).
Protein transmission was evaluated by collecting small samples of the filtrate and
bulk solutions after filtration of at least 500 μL to ensure equilibrium operation and to
clear the dead volume downstream of the membrane. Small samples of the bulk solution
were taken directly from the stirred cell (after clamping the filtrate port). All experiments
were performed at room temperature (22 ± 3 °C). The observed sieving coefficient was
calculated as:
[3.6]
where and are the protein concentrations in the filtrate and bulk solutions,
respectively. The stirred cell was carefully emptied, rinsed with DI water, and flushed
with at least 25 L/m2
of appropriate buffer between experiments. Dextran sieving
coefficients were evaluated using the same basic procedures, with the molecular weight
distribution of the dextrans in the feed and filtrate solutions analyzed using size exclusion
chromatography as discussed in Section 3.5.
38
3.4.4 Diafiltration
Diafiltration was performed using an Amicon 8200 stirred cell (Millipore
Corporation, Bedford, MA) with a 10 kDa Ultracel™ membrane to remove the dioxane
and any residual reactants formed during synthesis of the protein charge ladders. The
stirred cell was filled with the protein mixture and the feed reservoir was filled with
chilled DI water. The diafiltration was performed at a trans-membrane pressure of
approximately 6.9 kPa (1 psi) at room temperature (22 ± 3 °C) for at least four
diavolumes (defined as the ratio of the cumulative filtrate volume to the constant
retentate volume in the stirred cell).
3.4.5 Protein Fouling
Protein fouling experiments were performed with 5 g/L solutions of IgG. Limited
fouling experiments were also performed using 5 g/L solutions of α-lactalbumin,
lysozyme, and BSA. The membrane permeability was initially evaluated for each clean
membrane. The membrane was then soaked overnight in the protein solution, returned to
the stirred cell, rinsed with DI water, and the permeability re-evaluated. The membrane
was then flushed with at least 25 L/m2 of buffer at a pressure of 69 kPa (10 psi). The
stirred cell and feed reservoir were filled with the protein solution, the system was re-
pressurized to 69 kPa, and the filtrate flux was measured as a function of time for 60 min.
Filtrate samples were collected periodically for subsequent analysis, with bulk samples
collected directly from the stirred cell immediately before and after the experiment. After
39
completion of the ultrafiltration experiment, the stirred cell was carefully rinsed with DI
water and the buffer flux re-evaluated at 69 kPa. All experiments were performed at room
temperature (22 ± 3 °C).
The amount of protein adsorbed by the membrane in a static fouling system was
evaluated using the solution depletion method. A single membrane was placed in 10 mL
of a 5 g/L protein solution and allowed to soak overnight. The change in the protein
concentration in the solution was used to calculate the amount of protein adsorbed on the
membrane surface by a simple mass balance.
3.5 Size Exclusion Chromatography (SEC)
Dextran solutions were analyzed by size exclusion chromatography, also known
as gel permeation chromatography (GPC), to determine the concentration and molecular
weight distribution. An Agilent 1100 Series high performance liquid chromatography
system (Agilent Technologies, Palo Alto, CA) was used with a Superdex 200, 10/300
analytical column (13 μm particle size, 1 x 105 MW exclusion limit, from GE Healthcare,
Uppsala, Sweden). The mobile phase was a 10 mM Bis-Tris buffer at pH 7 containing
0.25 M KCl. The buffer was degassed before entering the system to avoid bubbles. The
column was initially equilibrated with a minimum of 2 column volumes of the mobile
phase at a flow rate of 0.3 mL/min. 25 μL samples of the dextran solution were then
injected by an autosampler immediately upstream of the guard column. The dextran
concentration in the exit stream was evaluated using a refractive index detector (Agilent
40
1100). Data collection was performed using ChemStation software version A.04.08
(Agilent Technologies, CA). Actual values were determined by comparison of the data
with that of known dextran standards.
3.6 Capillary electrophoresis
The concentration and net charge of each element of the charge ladders were
determined using a G1600A High-Performance Capillary Electrophoresis instrument
(Agilent Technologies, Palo Alto, CA) equipped with a dual polarity variable high
voltage DC power supply and variable wavelength UV-vis diode array detector.
Negatively charged fused silica capillaries (Agilent Technologies, G 1600-61211, Palo
Alto, CA) were used for the negatively charged α-lactalbumin, and positively charge
eCAP™ Amine capillaries (Beckman Coulter, Inc., 477431, Fullerton, CA) were used for
the positively charged lysozyme to minimize protein adsorption to the capillary wall.
Both capillaries had 50 μm inner diameters and were 65 cm in length (effective length of
approximately 55 cm). Protein detection was by UV absorbance at 214 nm. Mesityl oxide
(Fluka, 63940) was used as a neutral marker to evaluate the contribution of the electro-
osmotic flow. The capillaries were initially washed with 0.1 M NaOH for 10 min
followed by the running buffer (10 mM KCl in tris/glycine solution at pH 8.3) for an
additional 10 min. The eCAP™ Amine capillary was regenerated between runs using
amine regenerator solution (Beckman Coulter, Inc., 477433). 15-30 nL samples were
injected by application of a 3.5 kPa pressure for 25 s. Electrophoresis was performed at
an applied voltage around 25 kV. The electric field direction was chosen so that the
41
direction of the bulk flow was toward the outlet of the capillary. The current was kept
below 45 μA to minimize Joule heating. Electropherograms were recorded and analyzed
using 3D-CE ChemStation software (Version A.0903, Agilent Technologies, Palo Alto,
CA). Actual concentrations were evaluated by comparison with results for known protein
standards. Additional experimental details are available elsewhere (Ebersold and Zydney,
2004; Menon and Zydney, 1998).
3.7 X-ray Photoelectron Spectroscopy (XPS)
The extent of surface modification was estimated from the elemental composition
of the membrane as determined by X-ray photoelectron spectroscopy. The analysis was
performed using a Kratos Analytical Axis Ultra instrument (Kratos Analytical Inc.,
Chestnut Ridge, NY) available in the Materials Research Institute at The Pennsylvania
State University. The membrane was first flushed with deionized water, dried gently
using a Kimwipe, cut into small (approximately 12 mm x 5 mm) pieces using a razor
blade, and mounted on a sample platen. Data were obtained using monochromatic Al Kα
as the X-Ray source (1486.6 eV photons). The pressure in the analysis chamber was
Torr. The XPS data were analyzed using CasaXPS software (version 2.3.12Dev9)
by integrating the peak areas and applying the appropriate relative sensitivity factors to
account for the x-ray cross section and the transmission function of the spectrometer. All
binding energies were referred to the C1s peak at 285 eV.
42
Chapter 4
Fouling characteristics of zwitterionic membranes
Note: Most of the material presented in this Chapter was previously published (Hadidi
and Zydney, 2014)
4.1 Introduction
As discussed in Chapter 1, zwitterionic surfaces tend to adsorb very low amounts
of protein due to their high hydration capacity (via a combination of electrostatic and
hydrogen bonding interactions). The objective of the studies presented in this Chapter was
to obtain a fundamental understanding of the fouling behavior of a series of zwitterionic,
charged, and neutral membranes with nearly identical pore size / permeability. The
membranes were prepared by covalent attachment of small ligands to a base cellulose
(neutral) membrane, with the ligands specifically chosen to have the same effective size but
with different end functionality to generate an appropriate set of controls for the fouling
experiments.
4.2 Materials and Methods
The general procedures for the protein fouling experiments were described in
Chapter 3. Cellulose membranes were modified by activation with epichlorohydrin
43
followed by reaction with the appropriate ligand (Figures 3.3 and 3.4). Membranes were
characterized by both streaming potential measurements and XPS. Ultrafiltration
experiments were performed at constant transmembrane pressure using human serum
gamma globulin (IgG), bovine serum albumin (BSA), lysozyme, and α-lactalbumin as
model proteins. In addition, the amount of protein adsorbed on the membrane surface was
evaluated using the solution depletion method.
4.3 Results and Discussions
4.3.1 Membrane Modification
The extent of membrane modification was examined by X-ray photoelectron
spectroscopy (XPS). Figures 4.1 and 4.2 show XPS spectra for the upper surface
(approximately the upper 10 nm) of a zwitterionic, positive, acidic, hydrophilic,
hydrophobic, and sulfonic acid membrane. The spectra in Figure 4.1 were obtained
around the binding energy of nitrogen (398 eV). The nitrogen peak is absent in the base
cellulose (not shown) and in the membrane having the sulfonic acid modification but is
clearly visible in the other membranes. The double peak seen with the zwitterionic
membrane is likely associated with the presence of two distinct nitrogens, a primary
amine in the zwitterion and a secondary amine associated with the covalent linkage to the
membrane. Figure 4.2 shows the spectra around the sulfur peak (binding energy of 168
eV); in this case only the negatively charged membrane produced with the sulfonic acid
ligand showed a measurable peak in this range of binding energy as expected.
44
Figure 4.1 XPS spectra showing the nitrogen peak for all 100 kDa modified
membranes.
Figure 4.2 XPS spectra showing the sulfur peak for all 100 kDa modified membranes.
1000
1200
1400
1600
1800
2000
2200
2400
2600
385 390 395 400 405 410 415 420
Co
un
ts P
er
Seco
nd
Binding Energy (eV)
Hydrophobic
Positive
Acidic
Zwitterionic
Hydrophilic
Negative
100
200
300
400
500
600
700
150 155 160 165 170 175 180 185
Co
un
ts P
er
Seco
nd
Binding Energy (eV)
Negative
Acidic
Zwitterionic
Hydrophobic
hydrophilic
Positive
45
The atomic composition of the different membranes was determined from
quantitative analysis of the full XPS spectra based on a direct analysis of the peak area
associated with the C, O, N, and S atoms. The results are summarized in Table 4.1. The
nitrogen content was greatest for the zwitterionic and positively-charged membranes
since both of these membranes had two N in each ligand. The nitrogen content for the
acidic, hydrophobic, and hydrophilic membranes were similar, consistent with the
structure of these ligands (all with one N) and the use of the same epichlorohydrin
activation.
The degree of modification for each membrane was estimated from the atomic
fraction of nitrogen ( ) or sulfur as:
[4.1]
where f is the fraction of glucose rings in the base cellulose membrane that were
modified. The value of f is calculated assuming that all of the epichlorohydrin groups are
reacted with the ligand in the subsequent modification step. The parameters a, b, and d
are related to the atomic structure of the modified membrane: a is related to the number
of added N per ligand and b + df is the total number of atoms. For example, the
zwitterionic membrane modified with lysine has a=2 (accounting for two nitrogen atoms
in the two amine groups in lysine) and b=11 based on the number of carbon and oxygen
atoms in the base glucose ring (6 C and 5 O). The parameter d=14 based on the total
number of carbon, oxygen, and nitrogen atoms in the modified membrane (6 C, 2 O, and
2 N from the lysine along with 3 C and 1 O from the epichlorohydrin). This gives f =
0.025 for the zwitterionic membrane based on a nitrogen content of 0.43%.
46
The fraction of modified glucose rings for all membranes are summarized in
Table 4.1. All of the membranes prepared using the epichlorohydrin activation chemistry
had essentially the same degree of modification (f = 0.030 ± 0.004). The membrane
modified using the sulfonic acid ligand had a slightly lower degree of modification (f =
0.02) reflecting the difficulty in obtaining the same ligand density when using different
activation steps / reagents.
Table 4.1 Atomic composition (percent) and calculated degree of modification for
different 100 kDa modified membranes determined from XPS data.
Membrane f N Content C Content O Content S Content
Zwitterionic 0.025 ± 0.004 0.43 58.8 40.7 --
Positive 0.030 ± 0.004 0.52 60.0 39.4 --
Acidic 0.028 ± 0.004 0.25 60.5 39.2 --
Hydrophilic 0.026 ± 0.004 0.22 58.8 41.0 --
Hydrophobic 0.026 ± 0.004 0.23 60.6 39.1 --
Sulfonic acid 0.020 ± 0.004 -- 58.9 40.9 0.18
4.3.2 Membrane Surface Charge Characteristics
The effective surface charge of the different surface-modified ultrafiltration
membranes was evaluated from streaming potential measurements obtained with the fluid
flow directed through the membrane pores as described in Chapter 3. Figure 4.3 shows
47
typical experimental data for the measured streaming potential as a function of
transmembrane pressure ( ) for the zwitterionic version of the 100 kDa Ultracel™
membrane at different pH using 10 mM buffered KCl solution. The data were highly
linear at each pH with r2 values greater than 0.99. Repeat measurements for a given
membrane were highly reproducible with slopes within ±15%. The non-zero intercepts
are due to asymmetries in the Ag/AgCl electrodes and have no effect on the streaming
potential analysis (Burns and Zydney, 2000).
The data at pH 6, 7, and 8 have positive slopes, consistent with a net positive
charge on the membrane, while the slopes (and surface charge) are negative at pH 9 and
10. This behavior is due to deprotonation of the free amine functionality of the
zwitterionic membrane at high pH. The slope of the streaming potential versus pressure
data was used to calculate the apparent zeta potential of the membrane using the
Helmholtz-Smoluchowski equation (Equation 3.1):
[3.1]
with varying from +6.2 mV at pH 6 to -4.2 mV at pH 10.
48
Figure 4.3 Streaming potential data for 100 kDa Ultracel™ zwitterionic membrane in
10 mM buffered KCl solutions at several pH.
The charge characteristics of the zwitterionic membranes are examined in more
detail in Figure 4.4 which shows the measured values of the apparent zeta potential as a
function of the partial charge predicted using the pKa values for lysine: 9.06 for the
primary amine and 2.16 for the carboxylic acid (Brown, 1998). The pKa for the secondary
amine formed by the coupling of the lysine to the activated membrane was estimated as
8.8 based on the reported value for the primary amine in lysine (10.54) and the measured
difference in the pKa values of taurine (9.08) and TES (7.34) as reported by Rohani and
Zydney (Rohani and Zydney, 2012); the taurine and TES have analogous structures
except for the conversion of the primary amine in taurine into a secondary amine in TES.
The partial charge of the ligand was calculated using the Henderson-Hasselbach equation
as discussed in Chapter 2. The results are highly linear when plotted in this fashion with
r2 = 0.98, suggesting that the charge characteristics of the surface-grafted zwitterionic
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
5000 10000 15000 20000 25000 30000 35000
Str
eam
ing
po
ten
tial,
Ez (
mV
)
Transmembrane pressure, ΔP (Pa)
pH 6
pH 7
pH 8
pH 9
pH 10
49
membrane can be effectively described in terms of the protonation / deprotonation of the
acidic and basic components of the ligand.
Figure 4.4 Correlation between the apparent zeta potential and the calculated charge
based on the pKa values of the lysine ligand.
The results for the zwitterionic, positive, negative (sulfonic acid), hydrophilic, and
hydrophobic membranes at pH 7 are summarized in Table 4.2. The greatest positive
apparent zeta potential was obtained for the membrane modified with the diamine ligand
while the largest negative value was obtained with the sulfonic acid ligand as expected.
The zwitterionic (lysine-modified) membrane had a small positive charge due to the
secondary amine group formed by the covalent linkage to the cellulose membrane. This
secondary amine also provided the small positive charge on the hydrophilic and
hydrophobic membranes.
-5
-2.5
0
2.5
5
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25Ap
pare
nt
Zeta
Po
ten
tial,
𝜁𝑎𝑝𝑝 (m
V)
Calculated Fractional charge, Z
50
Table 4.2 The experimental values for the apparent zeta potential of surface-modified 100 kDa Ultracel™ membranes at pH 7 in 10 mM buffered KCl.
Membrane Zeta potential, (mV)
Zwitterionic 3.0 ± 0.1
Positively-charged 5.0 ± 0.1
Sulfonic Acid -9.4 ± 0.3
Hydrophilic 3.0 ± 0.1
Hydrophobic 4.3 ± 0.1
Acidic 0.5 ± 0.1
4.3.3 Dextran Ultrafiltration
To obtain additional insights into the sieving characteristics of the surface-
modified membranes, ultrafiltration experiments were performed using neutral dextrans.
The ultrafiltration was performed at relatively high ionic strength (150 mM) to minimize
electrostatic interactions. The dextran samples from the filtrate and feed were analyzed
by size exclusion chromatography, with the sieving coefficients calculated as the ratio of
the peak areas for narrow slices of the chromatogram (Mochizuki and Zydney, 1992).
Figure 5.5 shows results for the zwitterionic, hydrophilic, positive, and acidic membranes
over a wide range of dextran molecular weight. The sieving coefficients are plotted as a
function of the dextran radius, which was determined based on the retention time of
51
narrow molecular weight dextran standards using the correlation given by Equation [3.4].
The data for all four membranes are in fairly good agreement, indicating that these
membranes have very similar pore size characteristics. The dextran sieving coefficients
for the zwitterionic and hydrophilic membranes appear to be somewhat larger than those
for the acidic and positive membranes, which is consistent with the small differences in
hydraulic permeability for these membranes: for the zwitterionic and
hydrophilic compared to and for the acidic and positive
membranes, respectively.
Figure 4.5 Dextran sieving coefficients in 150 mM ionic strength at pH 7 using
different surface-modified membranes.
4.3.4 Static adsorption
The extent of static protein adsorption was evaluated using the solution depletion
method by measuring the change in protein concentration after incubation of the
membrane in a 5 g/L solution of serum IgG. Figure 5.1 shows results for the different
0
0.2
0.4
0.6
0.8
1
10 30 50 70 90
Sie
vin
g C
oeff
icie
nt,
So
Dextran Radius (Å)
Zwitterionic
Positive
Acidic
Hydrophilic
52
surface-modified membranes at pH 9 (i.e., where the IgG has a net negative charge),
plotted as the mass of protein adsorbed per unit cross-sectional area of the membrane.
The positively-charged membrane showed the greatest amount of protein adsorption,
consistent with the presence of an attractive electrostatic interaction. The zwitterionic and
negatively charged (sulfonic acid) membranes showed no measurable protein adsorption,
with the hydrophobic membrane showing approximately twice the amount of adsorption
as the hydrophilic surface.
Figure 4.6 Amount of protein adsorption from 5 g/L IgG at pH 9 on different modified
membranes.
Table 4.3 summarizes results for the membrane permeability before and after
overnight exposure to a 5 g/L serum IgG at different pH (in the absence of filtration). The
membranes used for these experiments were cut from adjacent areas of a single large
membrane sheet to ensure that the initial permeability of the unmodified membranes were
similar. The membrane permeability of the surface-modified membranes were also very
similar (variations of less than 10%), which is consistent with the use of the identical
0
200
400
600
800
1000
1200
Pro
tein
Ad
so
rpti
on
(μ
g/C
m2)
Zwitterionic
Hydrophilic
Hydrophobic
Positive
Sulfonic acid
53
activation chemistry coupled with the very similar size of the ligands. The permeability
of the negatively-charged membrane is slightly larger than that of the other membranes
due the smaller size of the sulfonic acid ligand (Figure 3.4). The permeability of the
zwitterionic membrane was largely unaffected by IgG adsorption, with less than a 12%
reduction in permeability under all conditions. The greatest fouling at pH 9 was seen with
the positively-charged membrane, consistent with the high amount of protein adsorption
due to the attractive electrostatic interactions under these conditions. The behavior at pH
5 was somewhat different since the IgG is positively-charged at this pH. Thus, the
negatively-charged membrane had the greatest reduction in permeability at pH 5.
Interestingly, the positively-charged membrane still showed more than a 30% reduction
in permeability at pH 5 even though the protein and membrane were like charged.
54
Table 4.3 Effect of protein adsorption on the permeability of the surface-modified 100 kDa Ultracel™ membranes.
Membrane
Clean membrane
Permeability, (m)
Pre-adsorbed
Permeability, (m)
%
Reduction
pH 5
Zwitterionic 3.6 0.4 3.4 0.3 6.9
Hydrophilic 3.8 0.4 3.6 0.4 5.1
Hydrophobic 3.5 0.4 3.1 0.3 13
Positive 3.5 0.4 2.4 0.2 32
Sulfonic Acid 3.8 0.4 2.0 0.2 48
pH 7
Zwitterionic 3.6 0.4 3.2 0.3 11
Hydrophilic 3.8 0.4 3.2 0.3 16
Hydrophobic 3.5 0.4 2.4 0.2 33
Positive 3.5 0.4 0.7 0.1 80
Sulfonic Acid 3.7 0.4 1.4 0.1 63
pH 9
Zwitterionic 3.3 0.3 3.0 0.3 11
Hydrophilic 3.3 0.3 2.0 0.2 39
Hydrophobic 3.3 0.3 1.8 0.2 47
Positive 3.3 0.3 1.3 0.1 61
Sulfonic Acid 3.5 0.4 2.3 0.2 30
4.3.5 IgG ultrafiltration
Protein ultrafiltration experiments were performed at a constant transmembrane
pressure of 69 kPa (10 psi). Figure 4.7 shows the flux data versus time for a single
experiment with a zwitterionic membrane, beginning with the buffer solution, followed
by the 5 g/L IgG, and then the buffer flux after the IgG filtration. The initial buffer flux
55
was 200 μm/s (720 L/m2/h), but this dropped to 11.8 μm/s with the start of the IgG
filtration due to the effects of concentration polarization and the osmotic pressure of the
highly retained IgG. The filtrate flux is nearly constant during the IgG ultrafiltration,
ranging from 11.8 to 11.2 µm/s, which suggests that there was relatively little fouling
over the course of the ultrafiltration process (beyond any immediate fouling upon
introduction of the protein solution at t = 10 min). At the end of the 1-hr filtration, the
stirred cell was then carefully emptied, refilled with buffer, and the flux re-evaluated at
the same transmembrane pressure. The buffer flux after the ultrafiltration was just
slightly greater than 160 µm/s, consistent with a small degree of fouling. Note that there
was no attempt made to clean the membrane at the end of the IgG filtration; the device
was simply emptied, refilled with a buffer solution, and then the stirrer was restarted.
Figure 4.7 Filtrate flux as a function of time during a typical fouling experiment for
the zwitterionic membrane using a 5 g/L IgG solution in 10 mM buffered
KCl at pH 7 and a constant pressure of 69 kPa (10 psi).
0
50
100
150
200
250
0 10 20 30 40 50 60 70 80
Flu
x (
µm
/s)
Time (min)
Buffer solution
Buffer solution
5 g/L IgG solution
56
The buffer flux and the initial filtrate flux with the 5 g/L IgG solution for the
different membranes are summarized in Table 4.4. The very low value of the buffer flux
for the positive membrane at pH 7 and 9 is due to protein adsorption; all of the
membranes examine in Table 4.4 were pre-soaked overnight in a 5 g/L solution of IgG at
the pH shown. The order of magnitude difference between the buffer and protein flux is
due to the large effect of concentration polarization. The zwitterionic membrane had the
highest protein flux at pH 7 and 9. The minimum in the flux for the zwitterionic
membrane occurred at pH 7, i.e., near the protein isoelectric point (pI), consistent with
previous experimental results (Fane et al., 1983). This is likely due primarily to the
increase in concentration polarization associated with the reduction in the protein
diffusion coefficient near the pI. The lowest flux at each pH was found with the
membrane that had a charge opposite to that of the protein. For example, the negatively-
charged membrane had the lowest protein flux at pH 5 (where the IgG was positively-
charged) while the positively-charged membrane had the lowest flux at pH 9.
57
Table 4.4 Buffer flux (after protein adsorption) and initial filtrate flux with a 5 g/L IgG solution for the different membranes at pH 5, 7, and 9.
pH 5 pH 7 pH 9
Buffer
flux,
( m/s)
Initial
protein
flux,
( m/s)
Buffer
flux,
( m/s)
Initial
protein
flux,
( m/s)
Buffer
flux,
( m/s)
Initial
protein
flux,
( m/s)
Zwitterionic 205 18.8 197 11.8 192 12.8
Hydrophilic 220 19.7 195 11.1 123 11.2
Positive 189 19.8 48 10.5 83.3 10.5
Sulfonic Acid 124 15.5 100 10.5 183 12.5
Figure 4.8 compares the measured values of the filtrate flux (top panel) and
filtrate concentration (bottom panel) during ultrafiltration of a 5 g/L IgG solution through
the different surface-modified membranes at pH 5. The flux for the zwitterionic and
positively-charged membranes are very similar, decreasing by approximately 15 % over
the course of the ultrafiltration. The flux data for the negatively-charged membrane are
quite different. The initial flux for the negative membrane was only 15.5 µm/s (compared
to 18.8 µm/s for the zwitterionic membrane), but the flux then increased slightly before
gradually decaying to a value that was very similar to that of the zwitterionic and
positively-charged membranes. The very different behavior for the negative membrane
may be related to a change in the properties of the pre-adsorbed protein on the surface of
this membrane at the start of the protein filtration. As seen in Tables 5.2 and 5.3, the
58
negatively-charged membrane had by far the greatest amount of protein adsorption at pH
5 (initial buffer flux of only 124 µm/s compared to 205 and 189 µm/s for the zwitterionic
and positively-charged membranes, respectively). Since the buffer permeability was
measured at pH 7, the adsorbed IgG would have been essentially neutral at the start of the
IgG filtration. Exposure of the pre-adsorbed IgG to the pH 5 buffer would lead to the
protonation of many of the ionizable amino acids, creating a net positive charge on the
protein and a corresponding expansion of the protein deposit and a reduction in the
hydraulic resistance to flow. This type of initial increase in flux was also seen in a fouling
experiment performed with the positively-charged membrane at pH 9, i.e., under
conditions where that membrane had a significant amount of pre-adsorbed protein.
Corresponding data for the observed sieving coefficients for IgG, defined as the
ratio of the filtrate to initial feed concentration, are shown in the bottom panel of Figure
4.8. The zwitterionic membrane had the largest initial sieving coefficient, which is
probably due to the smallest degree of protein adsorption. The IgG sieving coefficient for
the zwitterionic membrane remained relatively constant over the course of the
ultrafiltration; the slight decline in So is likely due to a combination of membrane fouling
and the small reduction in concentration polarization associated with the decrease in flux
over the course of the ultrafiltration. The greatest amount of IgG retention (smallest
sieving coefficients) was seen with the negatively-charged membrane, consistent with the
high degree of fouling seen with the negative membrane at pH 5, i.e., under conditions
where the protein and membrane are oppositely charged.
59
Figure 4.8 Filtrate flux (top panel) and filtrate concentration (bottom panel) for
ultrafiltration of a 5 g/L IgG solution in 10 mM buffered KCl at pH 5 at
a constant pressure of 69 kPa through the zwitterionic, positive, and
negative (sulfonic acid) membranes.
0
5
10
15
20
25
Flu
x (μ
m/s
)
Positive
Zwitterionic
Negative
0
0.02
0.04
0.06
0.08
0 10 20 30 40 50 60
Fil
trate
Co
ncen
trati
on
, C
f
Time
60
The results from the fouling experiments are summarized in Table 4.5 in terms of
the flux recovery ratio (F):
[4.2]
where and are the buffer flux of the clean membrane and the membrane after IgG
ultrafiltration, respectively. The flux recovery ratio (F) accounts for membrane fouling
under both static and dynamic flow conditions, with values of F close to one
corresponding to the absence of any significant fouling. The zwitterionic membrane had a
flux recovery ratio greater than 80% at all three pH, which is the best performance of any
of the membranes. This behavior is most pronounced at pH 9 where the zwitterionic
membrane had F = 0.82 while the best performance of the other membranes was F = 0.63
for the negatively charged membrane and only F = 0.36 for the positively charged
membrane. The hydrophilic membrane had very good performance at pH 5 (F = 0.84),
but this dropped to F = 0.57 at pH 9. The behavior of the positively- and negatively-
charged membranes were consistent with the differences in electrostatic interactions: the
performance of the negatively-charged membrane was best at pH 9 (F = 0.63), while the
performance of the positively-charged membrane was best at pH 5 (F = 0.58), in both
cases corresponding to conditions where the protein and membrane have the same
polarity (both negative or both positive, respectively).
61
Table 4.5 Flux recovery of different modified 100 kDa membranes after 1 hr ultrafiltration of an IgG solution at pH 5, 7, and 9.
pH Zwitterionic
Membrane
Hydrophilic
Membrane
Hydrophobic
Membranes
Positively-
charged
Membrane
Negatively-
charged
Membrane
5 89 84 63 58 45
7 80 74 57 26 35
9 82 57 49 36 63
Table 4.6 summarized the results for ultrafiltration of a 5 g/L IgG solution at pH 5
through the positive and zwitterionic membranes with and without the pre-adsorption
step. In both cases, the overall flux recovery was better for the membranes that were used
directly for the protein ultrafiltration, i.e., without exposing the membrane to IgG in the
pre-adsorption step. This effect was most pronounced for the positive membrane where
the flux recovery decreased from 72% to 58% when the membrane was pre-adsorbed
with IgG. The flux recovery for the zwitterionic membrane was 92% for the membrane
used directly for the protein ultrafiltration (without any pre-adsorption of the IgG).
62
Table 4.6 Initial buffer flux and flux recovery ratio for the zwitterionic and positively-
charged membranes after ultrafiltration of a 5 g/L IgG solution at pH 5 with
and without a pre-adsorption step.
Without adsorption With adsorption
Zwitterionic Positive Zwitterionic Positive
Initial Buffer Flux, (μm/s) 239 220 205 189
F (in %) 92 72 89 58
4.3.6 Protein ultrafiltration
Limited fouling experiments were also performed with other model proteins
having different size and surface charge characteristics to obtain additional insights into
the fouling resistance of the zwitterionic membranes. Experiments were performed
following the same basic procedures as used for the IgG ultrafiltration. This included
evaluating the clean membrane permeability, soaking the membranes overnight in a 5 g/L
solution of the protein, reevaluating the permeability, and then performing a constant
pressure ultrafiltration at 69 kPa for 1 hr. Table 4.7 summarizes the results in terms of the
flux recovery ratio, i.e., the ratio of the buffer flux after the protein ultrafiltration to that
of the clean membrane.
The zwitterionic membranes showed 92% and 81% flux recovery ratios after
ultrafiltration of lysozyme and α-lactalbumin at pH 7, respectively. These two proteins
have similar radius (1.6 nm, which is considerably smaller than the membrane pore size)
63
but very different surface charge characteristics, with the lysozyme being strongly
positively-charged while the α-lactalbumin has a significant negative charge at neutral pH
(Table 3.1). The lower flux recovery ratio after ultrafiltration of α-lactalbumin is
consistent with the weak attractive electrostatic interactions between the oppositely
charged α-lactalbumin and membrane at pH 7. The positively-charged membrane had a
similar flux recovery ratio as the zwitterionic membrane when used with lysozyme, but
the extent of irreversible fouling was much greater after ultrafiltration of the oppositely-
charged α-lactalbumin.
The data for BSA ultrafiltration were obtained at the protein isoelectric point (pH
4.7) and at relatively high solution ionic strength (150 mM), conditions that have
previously been shown to give high degrees of fouling (Fane et al., 1983). The
zwitterionic membrane showed F = 0.88 under these conditions compared to F = 0.76 for
the positively-charged membrane.
Table 4.7 Flux recovery of zwitterionic and positively-charged 100 kDa membranes
after 1 hr ultrafiltration of lysozyme and α-lactalbumin at pH 7 and BSA at pH 4.7.
Lysozyme α-Lactalbumin BSA
Zwitterionic 92 81 88
Positive 93 77 76
64
4.4 Conclusions
The data presented in this Chapter for the fouling behavior of the different surface
modified ultrafiltration membranes provide one of the most quantitative studies of the
performance of membranes with different surface functionalities (zwitterionic, positive,
negative, hydrophilic, and hydrophobic). The membranes were all prepared by covalent
modification of a base cellulose membrane with the same activation chemistry using a
series of ligands having essentially the same size/length but with different end-group
functionality. The resulting membranes had very similar hydraulic permeability and pore
size (dextran retention) but very different surface properties. This approach thus provides
an appropriate set of controls for understanding the effects of membrane surface
chemistry on the fouling characteristics.
The zwitterionic membrane displayed the greatest resistance to membrane fouling
over the full range of experimental conditions. This membrane showed negligible protein
adsorption in a static binding experiment, with the permeability of the protein-adsorbed
membrane decreasing only slightly compared to that of the pristine membrane.
Ultrafiltration of IgG caused a small additional reduction in permeability, but the flux
recovery for the zwitterionic membrane was greater than 80% at pH 5, 7, and 9
(corresponding to conditions where the protein was positively-charged, approximately
neutral, and negatively-charged). The hydrophilic membrane had a very similar flux
recovery value at pH 5, but this dropped to F < 0.6 when the IgG filtration was performed
at pH 9 while the zwitterionic membrane had F = 0.82 at this pH. The zwitterionic
membrane was also highly resistant to fouling of α-lactalbumin, lysozyme, and BSA,
65
with flux recovery ratios greater than 80% for all 3 proteins. This was true even for BSA
fouling at the protein isoelectric point, conditions that usually lead to extensive fouling.
Experimental data obtained with the positively- and negatively-charged
membranes clearly demonstrate that electrostatic interactions play an important role in
membrane fouling. The negatively-charged membrane showed its best performance
(greatest value of the flux recovery) at pH 9, where the IgG was also negatively charged.
In contrast, the positively-charged membrane had its best performance at pH 5 due to the
electrostatic repulsion between the protein and membrane under these conditions.
However, the largest flux recovery seen with the charged membranes was only F = 0.63,
which is well below the values obtained with the zwitterionic membrane.
Similarly, the results with the hydrophilic and hydrophobic membranes (generated
with the same ligands but with one having a hydroxyl group instead of a methyl group at
the end) show that hydrophilicity also has an impact on fouling, with the hydrophilic
membrane having a somewhat larger value of F over the entire pH range. However, the
flux recovery of the hydrophilic membrane remained below that for the zwitterionic
membrane at all three pH values. These results clearly demonstrate the very low fouling
characteristics of the zwitterionic surface, which could be very attractive for use in
ultrafiltration applications with highly fouling feed streams.
66
Chapter 5
Sieving characteristics of zwitterionic membranes
5.1 Introduction
As discussed in Chapter 1, it is now well-established that protein retention in
ultrafiltration is due to a combination of both protein size and electrostatic interactions
between the charged membrane and the charged protein. Rohani and Zydney (Rohani and
Zydney, 2012) recently extended these studies to zwitterionic membranes, performing
ultrafiltration experiments with acidic, basic, and neutral proteins. The protein sieving
coefficients were a strong function of solution conditions with the data for the different
proteins well correlated with the product of the surface charge densities of the protein and
membrane over a relatively wide range of conditions. However, the use of several different
proteins with different surface charge, charge distribution, and shape could easily have
obscured some of the key phenomena governing the behavior of these zwitterionic
membranes.
The primary objective of the studies presented in this Chapter was to use protein
charge ladders to quantitatively evaluate the effects of electrostatic interactions on protein
transport through a series of zwitterionic, charged, and neutral membranes prepared by
covalent attachment of small ligands to the base membrane. Protein charge ladders are
chemical derivatives of a protein differing by single charge groups, allowing the behavior of
67
a range of species with different charge, but essentially identical size / structure, to be
studied simultaneously in a single experiment.
5.2 Materials and Methods
The general procedures for evaluating the protein sieving characteristics were
described in Chapter 3. Membranes were modified by activation with epichlorohydrin
followed by reaction with the appropriate ligand (Figures 3.3 and 3.4). Membranes were
characterized by streaming potential measurements. Ultrafiltration experiments were
performed at constant transmembrane pressure using lysozyme and α-lactalbumin charge
ladders constructed by modification of the base protein as described in Chapter 3.
5.3 Results and Discussions
5.3.1 Membrane Characterization
As discussed in Chapters 3 and 4, the surface charge characteristics of the
different surface-modified ultrafiltration membranes were evaluated from streaming
potential measurements. The results for the zwitterionic, positively-charged, hydrophilic
and acidic membranes at pH 7 are summarized in Table 5.1. The greatest positive
apparent zeta potential was obtained for the membrane modified with the diamine ligand
(the positively-charged membrane). The zwitterionic (lysine-modified) and hydrophilic
68
membranes both had a small positive charge due to the protonation of the secondary
amine group formed by the covalent linkage to the cellulose membrane. The acidic
membrane was nearly neutral due to the positively-charged secondary amine and the
negatively charged carboxylic acid.
Table 5.1 Apparent zeta potential of surface-modified 100 kDa Ultracel™ membranes
at pH 7 in 10 mM buffered KCl.
Membrane Zeta potential, (mV)
Zwitterionic 3.0 ± 0.1
Positively-charged 5.0 ± 0.1
Hydrophilic 3.0 ± 0.1
Acidic 0.5 ± 0.1
5.3.2 Charge ladder Characterization
Figure 5.1 shows typical capillary electropherograms for 5 g/L solutions of the α-
lactalbumin (top panel) and lysozyme (bottom panel) charge ladders in a 10 mM ionic
strength tris/glycine buffer at pH 8.3. Transport in the capillary is dominated by the bulk
electroosmotic flow; the charged species migrate back against the electroosmotic flow
due to electrophoresis so that they pass the detector after the neutral marker. Thus, the
first peak in the electropherogram for α-lactalbumin represents the neutral marker
69
followed by the unmodified lactalbumin (which has the smallest negative charge) and
then the other protein derivatives, each of which has one less positive amine group (i.e.,
one more negative charge). 13 peaks or “rungs” are seen in the electropherogram,
corresponding to 0 to 12 acylated lysine groups. The electropherogram for the lysozyme
charge ladder begins with the most highly modified lysozyme species (with lowest net
positive charge at this pH), with the unmodified lysozyme eluting as the last peak. It is
just possible to make out 7 rungs in the charge ladder, consistent with the 6 lysine
residues in lysozyme.
70
Figure 5.1 Capillary electropherograms for α-lactalbumin (top panel) and lysozyme
(bottom panel) charge ladders in 10 mM tris/glycine buffer at pH 8.3.
The net charge for each of the protein variants was evaluated from the
electrophoretic mobility , which was determined from the migration times of the
neutral marker and the specific protein variant based on the location of the peak
maximum in the electropherogram as:
0
30
60
90
120
150
180
3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5
Ab
so
rban
ce (
mA
U)
Migration Time (min)
Neutral Marker
0
70
140
210
280
350
2.5 3 3.5 4 4.5 5
Ab
so
rban
ce (
mA
U)
Migration Time (min)
71
(
) [5.1]
where is the applied voltage, is effective capillary length, and and are the
migration times for the protein peak and the neutral marker, respectively. The net effective
charge was calculated from Equation 2.33 assuming that the protein is a uniformly charged
hard sphere (discussed in more detail in Chapter 2):
[2.33]
where is the protein radius, is the inverse Debye length (3.04 nm for the 10 mM buffer
solution used in the capillary electrophoresis), is the electron charge ,
and is Henry’s function (Henry, 1931) which accounts for the finite double layer
thickness (given by Equation 2.32).
The open symbols in Figure 5.2 show the calculated charge of each rung in the
charge ladder as determined from the capillary electrophoresis using Equations 5.1 and
2.33. Data for lysozyme were obtained using the positively-charged eCAP™ Amine
capillaries while those for α-lactalbumin were obtained with the negatively-charged fused
silica capillaries to minimize protein adsorption. The filled symbols in Figure 5.2 show
the calculated values of the net protein charge for the corresponding rungs calculated
from the amino acid composition by eliminating one lysine group for each rung. The
native lysozyme appears as peak 7 while the native α-lactalbumin is peak 1. The
calculated values of the lysozyme charge are in good agreement with the values from the
capillary electrophoresis; the deviations are somewhat larger for α-lactalbumin. The small
difference between the model calculations and the CE results could simply be due to the
72
approximations in the analysis, e.g., the assumption of a uniformly charged sphere;
although this might also reflect a small amount of ion binding in the CE experiments as
well as the effects of charge regulation (discussed below).
The net protein charge varies almost linearly with peak number, although the
change in charge between peaks is smaller than the one charge unit that would be
expected for the removal of one amine. For example, the net charge on α-lactalbumin
from CE decreases by 4.4 electronic charges as one goes from the unmodified protein
(Z=-4.3) to the protein with 6 reacted amine groups (Z=-8.7), which is less than the
expected reduction of 6 electronic charges. This behavior is due to charge regulation
effects associated with the change in protonation of the other charged amino acid residues
associated with the alteration in the local H+ concentration at the surface of the protein
caused by the change in net protein charge. For example, the increase in net negative
charge caused by the removal of one amine causes an increase in the local H+
concentration and a corresponding shift in the acid-base equilibrium leading to a small
increase in the degree of protonation. This is discussed in more detail elsewhere (Menon
and Zydney, 2000).
73
Figure 5.2 Net charge for the first seven peaks in the α-lactalbumin and lysozyme
charge ladders evaluated both from the electrophoretic mobility data
using 10 mM tris/glycine buffer at pH 8.3 (open symbols) and from the
amino acid composition (filled symbols).
5.3.3 Ultrafiltration Experiments
5.3.3.1 Protein Ultrafiltration
Figure 5.3 shows typical results for the observed sieving coefficients of several
“rungs” of the lysozyme charge ladder (i.e., individual peaks in the capillary
electropherogram) for the zwitterionic, hydrophilic, and positively-charged membranes.
The data are plotted as a function of the net protein charge determined from the amino
acid sequence; it was not possible to evaluate the charge at pH 7 from the electrophoretic
mobility due to the poor resolution of the capillary electrophoresis at this pH. The
-12
-10
-8
-6
-4
-2
0
2
4
6
8
0 1 2 3 4 5 6 7 8
Net
pro
tein
ch
arg
e, Z
Peak Number
Lysozyme-CE
α-lactalbumin-CE
α-lactalbumin-Amino acid
Lysozyem-Amino acid
74
observed sieving coefficient is defined as the ratio of the protein concentration in the
filtrate solution to that in the feed, with the concentration of each rung of the charge
ladder evaluated directly from the capillary electropherogram by numerical integration
under the peak (defined between adjacent local minima in the electropherogram). The
results for each membrane thus represent data obtained in a single ultrafiltration
experiment. The sieving coefficients for the zwitterionic, positively-charged, and
hydrophilic membranes decrease with increasing protein charge, which is a direct result
of the electrostatic repulsion between the positively-charged membranes and the
positively-charged lysozyme. The sieving coefficients for the zwitterionic and
hydrophilic membranes were very similar, consistent with the similar zeta potential for
these membranes, suggesting that the zwitterionic functionality at the end of the ligand
acts like a hydrophilic group. The sieving coefficients for the positively-charged
membrane display a greater slope, i.e., a greater reduction in sieving coefficient with
increasing protein charge, along with the a lower apparent intercept at zero protein
charge. This behavior is likely due to the larger positive charge (+5 mV compared to +3
mV for the zwitterionic and hydrophilic membranes) as well as the somewhat smaller
initial permeability (3.3 x 10-12
m compared to 3.6 x 10-12
m for the zwitterionic and
hydrophilic membranes), with the latter corresponding to a smaller effective pore size.
This smaller pore size is also seen in the slightly smaller values of the dextran sieving
coefficients for the positively-charged membrane (Figure 4.5).
75
Figure 5.3 Observed sieving coefficients for ultrafiltration of lysozyme charge ladder
at pH 7 through 100 kDa modified Ultracel™ membrane as a function of
net protein charge.
In order to obtain additional insights into the electrostatic interactions, the
observed sieving coefficient data for the different membranes are plotted in Figure 5.4 as
a function of the product of the dimensionless surface charge densities of the protein and
the membrane. This form is consistent with the theoretical analysis of the partitioning of
a charged sphere in a charged cylindrical pore (Smith and Deen, 1980) assuming that the
electrostatic interactions are dominated by the term arising from the interaction between
the electrical double layers of the protein and pore. The surface charge density for each
variant was calculated from the amino acid composition while the dimensionless
membrane surface charge density was evaluated from the apparent zeta potential
measurement at pH 7. The solid curves represent the theoretical calculations for
membranes with effective pore radii of 7.2 nm (blue and orange curves) and 9.2 nm (red
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5 6 7 8
Ob
serv
ed
Sie
vin
g C
oeff
icie
nt,
So
Net Protein Charge, Z
Zwitterionic
Hydrophilic
Positive
76
curve), determined by minimizing the sum of the squared residuals between the model
and data for the positively-charged / acidic membranes and for the zwitterionic /
hydrophilic membranes, respectively. The sieving coefficients decrease monotonically
with increasing values of the charge interaction parameter as expected. The small
difference between the blue and orange curves is due to the terms involving the square of
the protein charge and membrane charge in Equation 2.12. These terms are small relative
to the term involving the product of the protein and membrane charge, although they do
have a measurable effect on the sieving coefficient at large values of the protein charge.
The significant difference in the best fit values of the pore radius for the
positively-charged / acidic membranes and the zwitterionic / hydrophilic membranes is
surprising. The positively-charged / acidic membranes did have a slightly lower hydraulic
permeability, although the approximately 10% difference in permeabilities would
correspond to only a 5% difference in pore radius (assuming Poiseuille flow). One
possible explanation for this effect is the different structure of the charged ligands used to
construct the surface-modified membranes. The positively-charged and acidic
membranes both have charged groups at the far end of the ligand, towards the center of
the pore, and thus in close proximity to the protein. In contrast, the zwitterionic and
hydrophilic membranes have end groups that are electrically neutral, with the small
positive charge on these membranes arising from the secondary amine that is located
closer to the pore wall (and thus further away from the protein). Rohani et al. (Rohani et
al., 2010) reported the size of a diaminodecane ligand as 1.4 nm, which has the same
physical structure (but slightly larger size) as the diaminohexane ligand used to generate
the positively-charged membrane in this thesis. This is still somewhat smaller than the 2
77
nm difference in the best fit values of the pore size, but it is at least qualitatively
consistent with the experimental observations.
Figure 5.4 Observed sieving coefficients as a function of charge interaction parameter
(product of the dimensionless surface charge densities for the protein and
the membrane) for different membranes. Filled symbols represent the
experimental data with the solid curves representing the theoretical model.
5.4 Conclusions
Although previous studies of protein transport through semipermeable
ultrafiltration membranes clearly demonstrated the importance of electrostatic
interactions, there have been no quantitative studies comparing the sieving characteristics
of zwitterionic membranes with those of charged and neutral membranes with otherwise
78
similar properties. The experimental studies performed in this Chapter used protein
charge ladders consisting of a series of protein variants with the same 3-dimensional
structure and size but with different surface charge to probe the sieving characteristics of
the different membranes, allowing data to be taken over a range of protein charge in a
single experiment. This eliminates artifacts associated with differences in protein fouling
or variability in membrane properties between experiments.
Protein transmission through the zwitterionic, positively-charged, acidic, and
hydrophilic membranes was highly correlated with the product of the protein and
membrane charge, consistent with available theoretical models based on the partitioning
of a charged sphere in a charged cylindrical pore. The sieving coefficients for the
zwitterionic membrane were nearly identical to those for the hydrophilic membrane,
indicating that the zwitterionic group behaves like an uncharged hydrophilic (hydroxyl)
group in the context of protein transport. Interestingly, the model calculations suggest
that the zwitterionic and hydrophilic membranes have an effective pore size that is 2 nm
larger than that of the positively-charged and acidic membranes. This discrepancy
appears to be due to the different charge structure of the ligands, with the charge groups
in the positive and acidic membranes located at the end of the ligand (facing out into the
center of the pore) while the small positive charge on the zwitterionic and hydrophilic
membranes is associated with the secondary amine located much closer to the cellulose
surface. This suggests that these zwitterionic membranes might be able to provide the
enhanced permeability – selectivity characteristic of electrically charged membranes
while maintaining very low fouling characteristics due to the zwitterion at the outer
79
portion of the ligand. Additional experimental studies will be needed to confirm these
experimental results and provide further support for this physical picture.
80
Conclusion and future work
6.1 Introduction
The production of high value recombinant proteins requires robust, cost-effective,
and high-resolution separation methods that can provide high yield and purification of the
desired product. Ultrafiltration processes have remarkable potential to meet these needs
since they provide high throughput protein purification under mild conditions that will
not degrade or damage the biological product. One of the major challenges in many UF
processes is membrane fouling due to interactions between the proteins and membrane
surface. Several recent studies have shown that zwitterionic surfaces have low protein
adsorption characteristics due to their high degree of hydration associated with electrostatic
and hydrogen bonding interactions.
This thesis provides a quantitative study of the transport and fouling characteristics
of zwitterionic membranes in comparison with electrically charged and neutral membranes.
Data were obtained for a series of membranes prepared by covalent modification of a base
cellulose membrane using the same activation chemistry with a series of ligands having
essentially the same size/length but different end-group functionality. This approach thus
provided an appropriate set of controls for understanding the effect of membrane surface
chemistry on both fouling and protein transport. The following subsections summarize the
key experimental and theoretical results from the different parts of this thesis.
Recommendations for future work are also discussed.
81
6.2 Protein Transport through Surface Modified Membranes
Although previous studies of protein transport through semipermeable
zwitterionic membranes clearly demonstrated the importance of electrostatic interactions,
those data were obtained at different pH and with proteins having different surface charge
characteristics. The experimental studies performed in this thesis used protein charge
ladders consisting of a series of protein variants with the same 3-dimensional structure
and size but with different surface charge. Protein transmission though the zwitterionic,
positively-charged, acidic, and hydrophilic membranes was highly correlated with the
product of the protein and membrane charge, consistent with predictions of available
theoretical models based on the partitioning of a charged sphere in a charged cylindrical
pore. The very similar behavior of the zwitterionic and hydrophilic membranes suggests
that the zwitterionic ligand behaves as a hydrophilic (uncharged) functionality.
Interestingly, the model calculations indicate that the zwitterionic and hydrophilic
membranes have a much smaller effective pore size than the positive and acidic
membranes, even though all four membranes have fairly similar pore size based on the
measured values of the hydraulic permeability and dextran sieving coefficients. This is
likely due to the different structure of the ligands, with the positive charge on the
zwitterionic and hydrophilic membranes arising from the secondary amine that is located
near the polymer surface and thus a significant distance away from the protein within the
membrane pore.
82
6.3 Protein Fouling of Surface Modified Membranes
The data presented in Chapter 4 provided one of the most quantitative studies of
the fouling behavior of membranes with different surface functionalities (zwitterionic,
positive, negative, hydrophilic, and hydrophobic). The zwitterionic membrane displayed
the greatest resistance to membrane fouling over the full range of experimental
conditions. This membrane showed negligible protein adsorption in a static binding
experiment, with the permeability of the protein-adsorbed membrane decreasing only
slightly compared to that of the pristine membrane. Ultrafiltration of IgG caused a small
additional reduction in permeability, but the flux recovery for the zwitterionic membrane
was greater than 80% at pH 5, 7, and 9 (corresponding to conditions where the protein
was positively-charged, approximately neutral, and negatively-charged).
The data with the positively- and negatively-charged membranes clearly
demonstrated that electrostatic interactions play an important role in membrane fouling,
with the greatest fouling seen when the membrane and protein are oppositely charged.
Surface hydrophilicity also plays a role, with the hydrophilic membrane having a better
flux recovery than the corresponding hydrophobic membrane. However, the largest flux
recovery seen with the charged membranes was only F = 0.63, which is well below the
values obtained with the zwitterionic membrane. The very low fouling characteristics of
the zwitterionic surface make this membrane very attractive for use in protein
ultrafiltration.
83
6.4 Recommendations
The results presented in this thesis provide important insights into the protein
transport and fouling behavior of the zwitterionic membranes. However, there are a
number of important areas that would benefit from additional experimental and theoretical
investigations.
The experimental studies presented in this thesis were performed using a small
stirred cell. However, industrial ultrafiltration systems use tangential flow filtration (TFF)
modules that have much better mass transfer characteristics. The filtrate flux in these TFF
modules is likely to be more strongly influenced by membrane fouling due to the reduction
in concentration polarization effects; future experimental studies should be performed to
directly evaluate the flux and flux recovery for protein ultrafiltration in these tangential
flow modules. These studies should also examine the stability of the zwitterionic surfaces
upon exposure to strong cleaning solutions (like NaOH and NaOCl) and after multiple
ultrafiltration cycles.
It would also be very desirable to extend this work to other ultrafiltration
applications that require low fouling membranes. For example, ultrafiltration is used
extensively in water treatment applications, providing significant removal of natural
organic matter and viruses. The zwitterionic membranes produced in this thesis could be
very attractive in these water treatment applications if they are able to retain their very low
fouling characteristics. Other systems of interest would include DNA, surfactants, and
harvested cell culture fluid, all of which tend to be difficult to process using ultrafiltration
due to the high degree of fouling.
84
It would also be interesting to explore the further optimization of the ligand
structure for these zwitterionic UF membranes. For example, the zwitterionic ligand
could be attached to the membrane using a different linker, e.g., using a chemistry that
generates a fully neutral surface instead of the positively-charged surface produced with
the amine linkage examined in this thesis. It would also be possible to tune the properties
of the linker or the zwitterionic ligand, e.g., by optimizing the distance between the two
charged groups in the zwitterion or the length of the connector between the zwitterion and
the base membrane. It would also be interesting to perform experiments with “mixed-
charge” membranes generated by covalent attachment of a combination of separate
negatively-charged and positively-charged ligands to the base membrane. This would make
it possible to explore the effect of the charge distribution over the membrane surface on the
performance of the zwitterionic membrane.
The low fouling characteristics of zwitterionic surfaces are typically attributed to
the high degree of hydration of the zwitterion. More fundamental insights into the effects of
water dynamics, ligand chemistry/structure, and ligand distribution could potentially be
obtained using appropriate molecular dynamics simulations to evaluate the energy of
interaction between the protein and membrane surface. These simulations could then be
used to aid the design and selection of zwitterionic ligands for very low fouling
membranes.
85
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Appendix
Amino Acid Composition of Proteins
As discussed in Chapter 2, the protein surface charge density is determined by the
dissociation of the various ionizable amino acid residues on the surface of the protein.
The number and values of the various amino acids present in the proteins used in
this thesis are presented in Tables A.1 and A.2 below.
Table A.1 Number and values of charged amino acids in lysozyme (Sharma et
al., 2003)
Type
α-Amino 1 7.5
His 1 6.3
Arg 11 12.5
Lys 6 10.5
Glu 2 4.4
Asp 7 4
α-carboxyl 1 3.8
Tyr 3 9.6
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Table A.2 Number and values of charged amino acids α-lactalbumin (Molek,
2008).
Type
N-term 1 9.87
His 3 6.04
Arg 0 12.5
Lys 12 10.54
Glu 8 3.9
Asp 9 3.9
C-term 1 2.16
Tyr 4 10.3
The small differences in values in Tables A.1 and A.2, e.g., 6.3 and 6.04 for
histidine, reflect the range of pKa values for the various amino acids reported in the
literature and likely reflect small differences in ionization potential associated with the
specific local environment within the different proteins. All calculations in this thesis
were performed using the respective values in Tables A.1 and A.2 since those numbers
have been shown previously to properly describe the net charge for lysozyme and α-
lactalbumin, respectively.
