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INTRODUCTION
Microfiltration is used extensively for the clarification
and sterilization of many biological solutions, including
a variety of beverages, fermentation broths, and therapeutic proteins. Protein fouling is a critical factor in
many of these processes, resulting in a substantial reduction in membrane performance and a significant loss of
valuable product.
Recent studies have provided considerable insights
into the underlying fouling mechanisms as well as the
proper mathematical description of the flux decline in
these systems. Kelly et al. (1993) have shown that the
Correspondence to: A. L. Zydney
Contract grant sponsor: National Science Foundation
CCC 0006-3592/97/010091-10
solved protein. In order to avoid bacterial contamination, protein solutions were stored at 48C and used
within 48 h of preparation.
In some experiments, the protein solutions were prefiltered through 100,000 (100K) molecular weight cutoff (MWCO) Omega polyethersulfone ultrafiltration
membranes (Filtron Technology, Northborough, MA)
to remove protein aggregates and oligomers prior to
use in the microfiltration experiments. This prefiltration
was performed in a 64-mm-diameter stirred ultrafiltration cell (model 8200, Amicon, Beverly, MA) using nitrogen pressurization (36 psi) with the stirring speed
maintained between 90 and 120 rpm. The feed solution
reservoir and filtrate collection flasks were maintained
at 588C throughout the prefiltration to minimize the
possibility of protein denaturation. The prefiltration was
typically performed using protein solutions having
about twice the desired (final) concentration. The filtrate was then diluted with phosphate-buffered saline
(PBS) (prefiltered with a clean membrane of identical
MWCO) to obtain the desired bulk protein concentration.
Protein concentrations were determined by reacting
the proteins with copper to form a purple copper
protein complex. Two milliliters of Total Protein Reagent 541 (Sigma Chemical Co.) were added to a 4-mL
sample cuvette containing 800 mL of protein solution.
The solutions were mixed by gentle inversion and allowed to sit for 15 min. The absorbance was then measured at 540 nm using a Lambda 4B spectrophotometer,
with the actual protein concentrations determined using
appropriate calibration curves. Concentrations of BSA
and cys-BSA were determined by reaction with bromcresol green (Sigma Chemical Co.) with the absorbance
measured at 628 nm. The accuracy of the concentration
measurements was approximately 0.1 g/L.
Proteins were also analyzed using discontinuous pH
sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE). Gradient gels of approximately
3.520% acrylamide were cast in a model SE250 minigel
apparatus (Hoefer Scientific Instruments, San Francisco, CA). The upper and lower buffer reservoirs were
Protein
Sigma
catalog
number
Source
MW
(kD)
SSa
SHb
pI
BSA
Cys-BSA
Ovalbumin
Lysozyme
b-Lactoglobulin
a-Lactalbumin
Pepsin
Myoglobin
A7906
A0161
A5503
L6876
L6879
L5385
P6887
M0630
Bovine serum
Bovine serum
Chicken egg
Chicken egg
Cow milk
Cow milk
Pig stomach
Horse muscle
67
67
40
14
18
15
36
17
17
18
1
4
2
4
3
0
1
0
4
0
1
0
0
0
4.7
4.7
4.6
11.0
5.3
5.1
1.0
7.0
a
b
92
1 dJ
J dt
(1)
All derivatives were evaluated numerically using a central difference representation that accounted for the
Figure 1. Normalized filtrate flux (top panel) and rate of flux decline
(bottom panel) for the filtration of 2 g/L solutions of lysozyme, pepsin,
and cysteinylated BSA through 0.22-mm PVDF membranes at 14 kPa
and 600 rpm. Solid curves are model calculations.
nonuniform time spacing of the flux data and was accurate to O(Dt 2). The solid curves are model correlations
which are discussed in more detail subsequently. The
most rapid flux decline was obtained with pepsin, while
the least rapid decline was obtained with lysozyme. The
fouling time, defined as the time required for the filtrate
flux to decline to 20% of its initial value, ranged from
t 20 5 12 min for pepsin to t 20 5 54 min for cysBSA
and t 20 . 160 min for lysozyme. Repeat measurements
performed under identical experimental conditions
yielded filtrate flux data that were within 15% of the
values shown in Figure 1 throughout the filtration, with
the calculated values of the fouling parameters (discussed subsequently) varying by less than 30%. However, the averaged flux data for multiple runs could not
be used to evaluate the rate of flux decline (K) due to the
large errors introduced by the numerical differentiation.
Thus, all of the flux decline calculations shown in this
93
Figure 2. Normalized filtrate flux during the filtration of 100K-prefiltered solutions of lysozyme and cysteinylated BSA through 0.22-mm
PVDF membranes at 14 kPa and 600 rpm.
94
Figure 3. Normalized filtrate flux (top panel) and rate of flux decline
(bottom panel) for the filtration of 2 g/L solutions of b-lactoglobulin,
BSA, and ovalbumin through 0.22-mm PVDF membranes at 14 kPa
and 600 rpm. Solid curves are model calculations.
Figure 4. Normalized filtrate flux during the filtration of 100K-prefiltered solutions of BSA and ovalbumin through 0.22-mm PVDF membranes at 14 kPa and 600 rpm.
Torchinsky (1981), and Jocelyn (1972). Free sulfhydryls are known to participate in thiol oxidation and
thioldisulfide interchange reactions, both of which
result in the formation of intermolecular disulfide
linkages, which can in turn generate large aggregate
structures (Huggins et al., 1951; Liu et al., 1991). The
rate of these reactions is determined by the number,
accessibility, and reactivity of the free thiols and the
reacting disulfides.
Kelly and Zydney (1994) showed that the thiol
disulfide interchange reaction plays a critical role in the
fouling characteristics of BSA solutions; changes in pH
or the addition of chelating agents altered the rate of
these intermolecular reactions and significantly affected
the rate of flux decline. The results in Figures 1 and 3
for cys-BSA and BSA clearly confirm this behavior.
The cys-BSA is prepared directly from the normal BSA
product and is reported to differ from the normal BSA
only in the capping of the free sulfhydryl group on
the native protein. This cysteinylation eliminates the
reactivity of this sulfhydryl group, thereby eliminating
the increase in the rate of flux decline seen during protein microfiltration.
In order to further quantify the fouling behavior of
the different proteins, the filtrate flux data in Figures 1
and 3 were fit to the dual-mode fouling model developed
by Kelly and Zydney (1995). This model accounts for
two distinct mechanisms of protein fouling: pore
blockage associated with the deposition of large protein
aggregates and the chemical attachment of native (monomeric) protein to the growing deposit. The rate of
change in the number of open pores, Nopen , is expressed as
dNopen
5 2aC bAJ v,open 2 bC bAJv,open(N0 2 Nopen)
dt
(2)
where N0 is the total number of pores, A is the membrane area, C b is the bulk protein concentration, and
Jv,open is the filtrate flux through the open pores. The
parameter a is related to the rate of aggregate deposition as well as the efficiency of pore blockage. Here, a
is thus assumed to be proportional to the fraction of
the total protein present as aggregates in the bulk solution. The parameter b describes the rate of chemical
addition of the native protein to the growing deposit.
This rate is assumed to be proportional to both the
convective transport of native protein toward the membrane (Jv,openC b) and the number of aggregates already
on the membrane surface. Since the protein deposit
is permeable to the filtrate, Kelly and Zydney (1995)
evaluated the filtrate flow through the blocked pores
using a classical Darcys law model accounting for the
resistance of the membrane and protein layer in series.
The total flux through the open and blocked pores
was evaluated by appropriate integration of Eq. (2),
yielding (Kelly and Zydney, 1995)
95
Jv
5
J0
G DG
Jss
bN0
bN0
aA
112
1 2 exp J0C b
11
t
a
J0
N0
a
GD
bN0
bN0
aA
11
t
1 exp J0C b
a
N0
a
(3)
where Jss is the steady-state filtrate flux obtained at long
times. The instantaneous rate of flux decline (K) can
be calculated by direct substitution of Eq. (3) into Eq.
(1). Equation (3) is clearly phenomenological, and it
provides no insights into the actual location of the deposited protein on (or in) the membrane. However, this
model does provide a quantitative description of the
relative importance of the two fouling steps as characterized by the values of the parameters a and b.
The best fit values of the model parameters (a/N0 ,
b, and Jss) for the different protein filtration experiments
were evaluated by minimizing the sum of the squared
residuals between the experimental data for the filtrate
flux and the model [Eq. (3)]. The results are summarized
in Table II, with the error estimates determined directly
from the least-squares optimization. The model calculations (solid curves in both the upper and lower panels
of Figs. 1 and 3) were in excellent agreement with the
data for both Jv /J0 and K. The model properly captures
the initial increase in K, the rise through a maximum,
and the subsequent decline in K seen in Figure 3 for
b-lactoglobulin, BSA, and ovalbumin, and it properly
describes the continual decline in K seen in Figure 1
for lysozyme, pepsin, and cys-BSA.
All of the proteins possessing at least one free sulfhydryl (b-lactoglobulin, ovalbumin, and BSA) had relatively large values of the parameter b, which characterizes the rate of chemical addition of native protein to the
growing deposit. As discussed previously, this reaction
apparently occurs via the thioldisulfide interchange reaction and is thus dependent on the presence of a free
thiol group in the native protein structure. The actual
value of b should be related to the number, accessibility,
and reactivity of the free thiols and reactive disulfides.
This is discussed in more detail below.
In contrast to the results for b-lactoglobulin, ovalbumin, and BSA, the best fit value of the parameter b
for lysozyme was b 5 0.002 6 0.04 3 1023 g21, which
Table II. Best fit values of model parameters (a/N0), b, and Jss.
Protein
BSA
Cys-BSA
Ovalbumin
Lysozyme
b-Lactoglobulin
a-Lactalbumin
Pepsin
Myoglobin
96
a/N0
(g21)
b
(g21)
Jss
(mm/s)
q
(pH 7)
Surface area
(m2 3 1016)
6
6
6
6
6
6
6
6
2.6 6 0.02
0
3.9 6 0.6
0
5.0 6 0.9
0
0
8.5 6 0.8
11.6
11.2
11.7
15.2
10.8
15.9
7.3
7.0
218
218
210
19
211
28
214
0
1.63
1.63
0.97
0.53
0.94
0.53
0.74
0.47
0.4
0.7
2.3
0.3
4.7
0.7
5.8
1.0
0.02
0.03
0.1
0.03
0.4
0.01
0.2
0.03
of the greater number of free thiol groups in the ovalbumin structure compared to the single free sulfhydryl
in BSA.
The increased rate of flux decline seen at t . 60 min
in the experiments employing 100K-prefiltered solutions of BSA and ovalbumin (data from Fig. 4) was
apparently associated with the formation of new protein
aggregates during the actual filtration run. The formation of additional aggregates and oligomers was confirmed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis which provides a measure of size via the
rate of protein migration though a polyacrylamide gel.
SDSPAGE showed a small increase in the number of
protein dimers and higher molecular weight oligomers
in the bulk protein solutions in the stirred cell over the
course of the ovalbumin and BSA filtration. The slow
onset of fouling seen for BSA and ovalbumin in Figure
4 is likely due to the slow kinetics of the intermolecular
thioldisulfide interchange reactions that are likely the
cause of aggregate formation for these proteins. Note
that cys-BSA and lysozyme, both of which lack any free
sulfhydryls, showed essentially no flux decline after the
removal of the protein aggregates (Fig. 2).
The data presented in Figure 3 suggest that the filtrate
flux approaches a quasi-steady value at long times. This
behavior is similar to the critical flux concept presented
by Bacchin et al. (1995), with Jss representing the filtrate
flux below which there is no longer any significant fouling. Palecek and Zydney (1994) hypothesized that this
quasi-steady flux is attained when the hydrodynamic
drag force on the proteins associated with the convective
filtrate flow toward the membrane is no longer sufficient
to overcome the repulsive (primarily electrostatic) interactions that exist between the native (bulk) protein and
the protein deposit on the membrane surface. As long as
the convective drag force is greater than the electrostatic
repulsion, proteins continue to add to the deposit and
the flux continues to decline. This model assumes that
fouling is governed primarily by the formation of a protein layer on the upstream surface of the membrane.
Internal (pore) adsorption is implicitly assumed to occur
quite rapidly upon exposure of the membrane to the
protein solution and thus has little direct effect on the
magnitude of the quasi-steady flux.
Palecek and Zydney (1994) estimated the electrostatic repulsive interaction using an expression for the
force between two charged spheres, while the hydrodynamic force was evaluated using the Stokes drag law.
The quasi-steady flux was then determined by equating
these forces, yielding (Palecek and Zydney, 1994)
Jv 5 JpI 1 vs 2 exp(2kh)
(4)
97
Figure 6. Quasi-steady filtrate flux obtained during protein microfiltration over a range of pH values as a function of the square of the
protein surface charge density. Calculations are described in the text.
98
intermolecular interaction (Gordon, 1971). Pepsin is unstable at pH values above 6 (Bovey and Yanan, 1960),
and lysozyme is known to form dimers at pH above 5
(Sophianopoulos and Van Holde, 1964).
Protein aggregation can also occur due to pumping
in cross-flow filtration systems (Chandavarkar, 1990;
Meireles et al., 1991), although this type of aggregation
was unlikely to be significant in the nitrogen-pressurized
stirred cell employed in this study. It is also likely that
the chemistry and morphology of the membrane, as well
as the hydrodynamic characteristics of the membrane
module, can affect the rate and extent of aggregate
deposition. Additional research is needed to determine
the actual mechanisms of aggregate formation for different proteins and to develop appropriate strategies for
eliminating the formation and deposition of these large
aggregate structures during protein filtration.
As discussed in the text, the greater rate of chemical
addition of ovalbumin to the growing deposit relative
to BSA may have been due to the presence of four free
sulfhydryls in ovalbumin compared to the single free
sulfhydryl in BSA. However, it was not possible to actually correlate the value of b with the number of free
sulfhydryls. For example, the largest value of b was
obtained with b-lactoglobulin, which only has a single
free sulfhydryl group. The actual rate of the intermolecular thiol-disulfide interchange reaction is dependent
on the reactivity (and not just the number) of the free
sulfhydryls, as well as the nature of the existing disulfide
bonds and the steric accessibility of the reacting moieties. A much more complete analysis of the detailed
protein structure in the vicinity of the free sulfhydryls
would be required to actually predict the value of b for
a given protein. In addition, the reactivity of the free
sulfhydryls can be strongly affected by the solution
chemistry. Kelly and Zydney (1994) have shown that
the presence of trace amounts of copper significantly
increased the rate of flux decline for BSA through the
catalytic activity of this divalent metal cation for these
sulfhydryl-mediated reactions. Metal chelators, which
effectively remove metal cations from solution, have
been successfully used to reduce the rate of fouling
(Kelly and Zydney, 1994).
Although this study provides the clearest demonstration of the importance of the free sulfhydryl group in
the fouling characteristics of different proteins, Lee and
Merson (1976) previously demonstrated that the rate
and extent of flux decline during the ultrafiltration of
cottage cheese whey could be significantly reduced by
addition of very small amounts of N-ethylmaleimide.
This chemical agent is known to block the free thiol
groups of both b-lactoglobulin and BSA (Lee and Merson, 1976), which together comprise over 50% of the
whey proteins. The ultrafiltration flux increased by
nearly 50% upon addition of 0.0015 M N-ethylmaleimide and by more than 75% upon addition of 0.0015 M
N-ethylmaleimide and 0.2 M CaCl2 . The dramatic effect
of the N-ethylmaleimide on the flux is completely consistent with the data obtained in this study for cys-BSA;
the cysteinylation eliminated the reactivity of the free
sulfhydryl, thereby dramatically reducing the rate of flux
decline. A more detailed understanding of the chemistry
of these intermolecular interactions, and their relationship to the underlying protein structure and properties,
will hopefully provide new strategies for the effective
control of protein fouling in both ultrafiltration and
microfiltration.
References
Bacchin, P., Aimar, P., Sanchez, V. 1995. Model for colloidal fouling
of membranes. AIChE J. 41: 368376.
Belfort, G., Davis, R H., Zydney, A. L. 1994. The behavior of suspensions and macromolecular solutions in crossflow microfiltration. J.
Membrane Sci. 96: 158.
Bollag, D. M., Edelstein, S. J. 1991. Protein methods. Wiley-Liss,
New York.
Bovey, F. A., Yanari, S. S. 1960. Pepsin, pp. 6392. In: P. D. Boyer,
H. Lardy, and K. Myrback (eds.), The enzymes. Academic, New
York.
Cecil, R., McPhee, J. R. 1959. The sulfur chemistry of proteins, pp.
255389. In: C. B. Anfinsen, Jr., M. L. Anson, K. Bailey, and J. T.
Edsall (eds.), Advances in protein chemistry, vol. 14. Academic,
New York.
Chandavarkar, A. S. 1990. Dynamics of fouling of microporous membranes by proteins. Ph.D. Thesis, Massachusetts Institute of Technology, Cambridge, MA.
Foster, J. F. 1977. Some aspects of the structure and conformational
properties of serum albumin, pp. 5384. In: V. M. Rosenoer,
M. Oratz, and M. A. Rothschild (eds.), Albumin structure, function,
and uses. Pergamon, Oxford.
Gordon, W. G. 1971. In: H. A. McKenzie (ed.), Milk proteins: Chemistry and molecular biology, vol. 2. Academic, New York.
Hlavacek, M., Bouchet, F. 1993. Constant flowrate blocking laws and
an example of their application to dead-end microfiltration of protein solutions. J. Membrane Sci. 82: 285295.
Huggins, C., Tapley, D. F., Jensen, E. V. 1951. Sulfhydryl-disulphide
relationships in the induction of gels in proteins by urea. Nature
167: 592593.
Jocelyn, P. C. 1972. Biochemistry of the SH group. Academic, London.
Jonsson, G., Pradanos, P., Hernandez, A. 1996. Fouling phenomena
in microporous membranes. Flux decline kinetics and structural
modifications. J. Membrane Sci. 112: 171184.
Kelly, S. T., Zydney, A. L. 1994. Effect of thiol-disulfide interchange
reactions on albumin fouling during membrane microfiltration. Biotechnol. Bioeng. 44: 972982.
Kelly, S. T., Zydney, A. L. 1995. Fouling mechanisms during BSA
microfiltration. J. Membrane Sci. 107: 115127.
Kelly, S. T., Opong, W. S., Zydney, A. L. 1993. The influence of
protein aggregates on the fouling of microfiltration membranes
during stirred cell filtration. J. Membrane Sci. 80: 175187.
Kim, K. J., Chen, V., Fane, A. G. 1993. Some factors determining
protein aggregation during ultrafiltration. Biotechnol. Bioeng. 42:
260265.
Lee, D. N., Merson, R. L. 1976. Chemical treatments of cottage cheese
whey to reduce fouling of ultrafiltration membranes. J. Food Sci.
41: 778786.
Liu, W. R., Langer, R., Klibanov, A. M. 1991. Moisture-induced aggregation of lyophilized proteins in the solid state. Biotechnol. Bioeng.
37: 177184.
Meireles, M., Aimar, P., Sanchez, V. 1991. Albumin denaturation
during ultrafiltration: Effects of operating conditions and conse-
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