Fractionation of BSA and lysozyme using ultrafiltration: effect of pH and membrane pretreatment

journal of MEMBRANE SCIENCE

ELSEVIER

Journal of Membrane Science 139 (19981 17-28

Fractionation of BSA and lysozyme using ultrafiltration: effect of pH and membrane pretreatment R. Ghosh, Z.F. Cui* Department of Engineering Science, University of O.~jbrd, Parks Road, O.~;ford OXI 3P.I, UK

Received 3 March 1997: received in revised fi)rm 4 September 1997: accepted 6 September 19~)7

Abstract Selective transmission of a solute through membranes proves to be a challenge in ultrafiltration processes. This is because the transport of a solute through an ultrafiltration membrane does not depend on size alone, but on several other factors such as solute-solute and solute-membrane interactions. By manipulating physicochemical parameters and process variables (eg. pH, ionic strength, concentration of solute, etc.) and by membrane modification, it is possible to enhance the transmission of a particular solute and thus enhance fractionation of solutes. In this paper, the effect of pH on fractionation of BSA and lysozyme by ultrafiltration through 50 kDa MWCO (molecular weight cut off) polysulfone membrane has been examined. It was found that the selectivity of solute separation for dilute mixtures of BSA and lysozyme was very much pH dependent and varied from 3.3 at pH 5.2 to 220.0 at pH 8.8. However, at a higher feed concentration, the transmission of lysozyme through polysulfone membrane decreases quite dramatically resulting in lower throughput of product. An attempt has been made to enhance the transmission of lysozyme through the polysulfone ultrafiltration membrane by pretreating the surface of the membrane by adsorption of another protein, myoglobin. An increase in lysozyme transmission of up to 63% with respect to native membrane was observed. The stability of this pretreatment and its effect on permeate flux have been examined. The pretreated membrane was used to fractionate BSA/lysozyme mixtures. Even at higher feed concentration, enhanced fractionation with respect to native membrane was observed due to highly enhanced transmission of lysozyme through the pretreated membrane. ~'", 1998 Elsevier Science B.V. Keywords: Ultrafiltration; Protein fractionation; Pretreatment; Lysozyme: BSA: Polysulfone membrane

1. I n t r o d u c t i o n Ultrafiltration is gradually e m e r g i n g as a powerful bioseparation process for purification and polishing of bioproducts such as therapeutic drugs, enzymes, hormones, antibodies, etc. Earlier, applications of ultrafiltration were limited to operations such as *Corresponding author. Tel.: +44 1865 273118; fax: +44 1865 273905: e-mail: [email protected] 0376-7388/98/$19.00 ~i, 1998 Elsevier Science B.V. All rights reserved. Pll S0376-73 88(97)00236-6

diafiltration and concentration, where the solutes to be separated had more than ten told difference in molecular weight. In such operations, size was the sole criteria for separation. However, it is now evident that ultrafiltration is a much more complex process, and that size is not the only criteria for solute separation. It is in fact possible to separate m a c r o m o l e c u l a r solutes having comparable molecular weights [1-13], and a lot of interest has been generated in this area of m e m b r a n e research. The inherently high throughput

18

R. Ghosh, Z.E Cui/Journal of Membrane Science 139 (1998) 17-28

of ultrafiltration makes it ideally suited for process scale bioseparation of proteins, The two major parameters to be considered in any ultrafiltration process are permeate flux J, and solute transmission. Solute transmission is usually expressed in terms of the percentage transmission observed O-ob), which is also referred to as the apparent sieving coefficient (Sa). Cp "Fob X 100 Cb For fractionation of a binary mixture of solutes, it is desirable to achieve maximum transmission of one solute and minimum transmission of the other. Efficiency of solute fractionation for a binary mixture is expressed in terms of the selectivity (~b): Tobl 7-ob2

By suitably manipulating these parameters, it is possible to obtain conditions at which there will be maximum transmission of the solute desirable in the permeate, and minimum transmission of the solute desirable in the retentate, with a specifically selected membrane. Balakrishnan et al. [1] have discussed the fractionation of lysozyme/ovalbumin and lysozyme/myoglobin mixtures using 100 kDa MWCO (molecular weight cut Off) hydrophilic polyacrylonitrile membrane in a vortex flow ultrafilter. Van Eijndhoven et al. [2] have reported selectivity values of more than 70 for a haemoglobin/BSA system being fractionated using 100 kDa MWCO Omega (polyethersulfone) membrane in a stirred cell ultrafilter. Saksena et al. [4] have studied the transport behaviour of IgG and BSA as single component solution and as binary mixtures using 100 kDa and 300 kDa MWCO polyethersulfone

where 7-ob1 is the observed percentage transmission of solute 1 and 7ob 2 is the observed percentage transmission of solute 2. Another parameter which may be useful in quantifying solute transport through a membrane is the solute flux (Ji). The ratio of the number of solute molecules going through a membrane for a binary mixture is the molar selectivity (~M): Jil ~bM = - Ji2 where Ji 1 is the solute flux of solute 1 and Ji 2 is the solute flux of solute 2. The membrane surface acts as the interface between the solution phase and the solid membrane phase. Physicochemical interactions occur between the membrane and the solutes and these interactions can be electrostatic, hydrophobic or due to charge transfer. As a result, the transmission of a solute through a specific membrane can be a strong function of physicochemical parameters such as pH and ionic strength, and also operational parameters such as transmembrane pressure, system hydrodynamics, etc. These interactions are also dependent on the solute concentration and hence the transmission is also affected by concentration polarization and membrane fouling. It is normally observed that proteins have their highest transmission at their isoelectric point, and lower transmissions further away from it, on both side [2,4,8]. It is also observed that the pH effect is more pronounced at lower ionic strengths [2].

membranes in a stirred cell module. They have investigated the effects of pH and ionic strength on fractionation. Selectivity values ranging from 2 to 50, depending on operating and physicochemical conditions were reported. Nel et al. [5], Zhang et al. [7] and Higuchi et al. [13] have also described processes for fractionation of IgG/BSA mixtures. Other reports on fractionation of macromolecules of comparable size have described the separation PEG and albumin [6], cytochrome-c and human albumin [8], ribonuclease and haemoglobin [8], and myoglobin and BSA [10]. Ingham et al. [9] have discussed the separation of BSA and lysozyme using Amicon PM-30 membrane. The separation was carried out in a diafiltration mode in which lysozyme was washed out of the system whileBSAwasquantitativelyretained.Theeffectofsalt concentration and BSA-lysozymeinteraction onthe rate oflysozymewashoutwasdiscussed.Therateofwashout was lower in the presence of BSA. Also, in the absence of salt, total rejection of lysozyme was reported. Iritani et al. [3] have reported the fractionation of lysozyme and BSA using a 30 kDa polysulfone membrane. The membrane was assumed to be almost completely retentive for BSA but permeable for lysozyme. The study was meant to demonstrate that electrostatic interactions between dissimilar molecules may control the solute rejection and the filtration rate in upward dead-end ultrafiltration of binary protein mixtures. The concentration of BSA was found to have a very strong influence on the transmission of

=

-

-

R. Ghosh, ZF. Cui/Journal qf Membrane Science 139 (1998) 1~28

lysozyme. At higher concentrations of BSA, greater rejection of lysozyme was observed. Studies were carried out at two different pH values and different ionic strengths. At pH4.0, withhigh salt concentration (300 mol m 3), the transmission of lysozyme was moderate. When no salt is present, a very low transmission was observed. At pH 7.0, with high salt concentration (600 m o l m 3), the transmission of lysozyme ranged from high to moderate, with a very sharp decline with time. When no salt is present, low transmission was observed. A 30 kDa membrane may not be ideally suited for fractionation of BSA and lysozyme, as lysozyme transmission on its own is low and it is quite obviously further decreased by the presence of BSA. Millesime et al. [11] have reported the fractionation of BSA and lysozyme using unmodified and modified inorganic membranes. They have investigated the effect of ionic strength on fractionation using both types of membranes. At low ionic strength, a selectivity of 10 was observed irrespective of the type of membrane used. However, at moderate and high salt concentrations, selectivity with modified membrane went down to 6 and was even lower with the unmodified membrane, As protein fractionation is affected by interactions between the protein molecules and the membrane, the selectivity may be improved by manipulating the surface characteristics on the membrane. Surface pretreatment may be the easiest and the most economical way of altering the surface properties of commercially available membranes. The use of surface modification or pretreatment of ultrafiltration and reverse osmosis membranes using chemical reagents and irradiation has been reported by several authors, e.g. Refs. [11-18]. There also exist a few reports, e.g. Refs. [2,4,10,18], on modification and pretreatment of membranes using proteins. However, the main theme in all these reports has been fouling control to prevent flux decline with time. Only a few reports, e.g. Refs. [10,18], describe the effects of surface modification by protein adsorption on solute transmission, Protein adsorption on hydrophobic membranes can be used as an effective method of pretreatment as the adsorption is most likely irreversible under normal operating conditions. The protein molecules which adsorb first are not displaced by other protein molecules during ultrafiltration, and this

19

results in a relatively stable pretreated membrane. Nakatsuka et al. [10] have demonstrated that higher transmission of myoglobin was obtained when filtering a solution of myoglobin through polysulfone membranes pretreated with myoglobin than when using a 'clean' membrane. Various different modes of pretreatmentie, passive adsorption, ultrafiltrationof protein solution, and combination of these were investigated. Ehsani et al. [18] have demonstrated that modified polysulfone membranes treated with either BSA or myoglobin or a mixture of them, show improved selectivity in the fractionation of BSA and myoglobin. They also indicated that the pH value affects myoglobin transmission significantly in such operations. In the present investigation, an attempt is made to study the effect of pH on the selectivity of fractionation of BSA (MW 67,000) and lysozyme (MW 14,100) by ultrafiltration through a 50 kDa polysulfone merebrane. The effect of pH is studied at low and high feed concentrations and the differences in efficiency of separation are studied. A further attempt is made to enhance the transmission of lysozyme through the polysulfone membrane by pretreatment of the membrane by adsorption of another protein myoglobin (MW 17,000). The effect of this membrane pretreatment on fractionation of BSA and lysozyme is examined.

2. Experiment 2.1. Materials

Lysozyme (from chicken egg white, MW 14,100, p l = 11.0), BSA (MW 67,000, p i - 4 . 9 ) and myoglobin (from horse heart, MW 17,000, pl=7.0) were putchased from Sigma. All test solutions were prepared using bi-distilled water microfiltered through 0.1 micron membrane (Whatman). Protein solutions were prepared by dissolving them to the appropriate weight percentages in sodium phosphate buffer (20 raM). All protein solutions were prefiltered using 0.1 micron microfiltration membranes, before the ultrafiltration experiments to remove aggregates, although no significant amounts of aggregates were detected in the experiments. Polysulfone membrane sheets (type GR51PP) having 50 kDa molecular weight cut off (MWCO), and reported to have an isoelectric point

R. Ghosh, Z E Cui/Journal of Membrane Science 139 (1998) 17-28

20

around 4.0 were purchased from Dow Danmark A/S and 25 mm circular discs were prepared in the laboratory to suit the filtration units,

2.2. Pretreatment of the membrane Pretreatment of polysulfone membrane with myoglobin was carried out at five different pH values, i.e. 5.2, 6.0, 7.0, 8.0 and 8.8, corresponding to the different operational pH values for the ultrafiltration experiments. The membranes were soaked at ambient temperature for 12 h, in 0.5 kg m -3 myoglobin solution, prepared in respective buffer. After soaking, the membranes were rinsed using buffer, and then compacted by filtering the respective buffer at 20 kPa transmembrane pressure for 20 min. The filtrate samples obtained during compaction were analysed to monitor the presence of rnyoglobin in order to access the stability of the pretreatment method,

2.3. Apparatus and procedure The experimental set-up is shown in Fig. 1. Purpose built stirred cells having working volume of 16 ml were designed and fabricated. These could be fitted with the 25 mm diameter membrane discs. Whatman (MS 500) magnetic stirrers were used to drive the stirrer bars, and for all the experiments, the stirring rate was kept fixed at 1400 rpm (+10 rpm) which was

monitored using a digital photo tachometer (RS 1635348). Nitrogen gas was used to pressurize the system and to maintain the transmembrane pressure. Permeate and retentate samples were periodically collected during the ultrafiltration runs to measure the solute concentrations using FPLC (Fast Protein Liquid Chromatography, Pharmacia), following a protocol based on MonoQ anion exchange column which was developed for analysis of the proteins. Unless otherwise stated, ultrafiltration experiments were run for 20 rain and up to 4 samples of retentate and permeate each, were collected for every run. Flux readings were taken by weighing out samples collected over a certain period of time, using an electronic balance. All experiments were run under ambient temperature conditions in the lab (25°C). The effect of pH was studied at five different pH values, ie. 5.2, 6.0, 7.0, 8.0 and 8.8. In each case, 20 mM sodium phosphate buffer was used and the pH was adjusted using NaOH. As fresh membrane discs, either native or pretreated were used for each experiment, it is important to ascertain that all the discs have hydraulic permeability values within a narrow range. The average pure water flux of the native membranes was about 5.0x 10 3 kg m 2 s-1 at 20 kPa transmembrane pressure. The variation of flux with different membrane discs was within 4-7.5% of the average value.

3. R e s u l t s a n d d i s c u s s i o n Pressure regulator

3.1. Single component filtration of lysozyme and BSA with native membrane

Pressure gauge

u

~ t ~

Nitrogen cylinder

F~.d reservoir

Sample

collection

tiffedeell I ultratiltration [mod~e M~b~a.e } -Permeate -~" Ii I ' ~ ~ Magnetic steel"

!

I

[ I Electronicb~ec Fig. 1. Experimental set-up,

The effect of pH on the permeate flux and transmission of lysozyme and BSA when filtered as single component solutions through 50 kDa MWCO polysulfone membrane was studied. The lysozyme and BSA concentration used in each case was 0.5 kg m -3. Flux and transmission data are shown in Fig. 2. The results clearly show that transmission of both lysozyme and BSA depend on the pH of the solution. The results are also consistent with the fact that highest protein transmission takes place near the pl of that protein. Polysulfone membrane has an isoelectric point around 4.0 and so at all the different pH values examined, the membrane will be mildly negatively charged. Since the pl of lysozyme is 11.0, it will

21

R. Ghosh, Z.E Cui/Journal of Membrane Science 139(1998) 17-28 0.004,

0.0035

100

~

80

/

) =

./

"~

0.0025

/

/

= ~

/

/

TMP: 20 kPa

J

Feed:.) o.s kg _

.

g

BSA

80

)o .kg

Stirrerspeed::1400rpm Buffer: 20 mM phosphate

mE ¢

~

a.

'= ."-

40

0.002

0.0015

0.001

~

(

l

y

s

o

z

y

0

Flux (BSA) Flux (lysozyme) Transmission(BSA) m e )

20

I

I

I

I

I

T

I

5.5

6

6.5

7

7.5

8

8.5

"¢

0

9

pH

Fig. 2. Flux and transmission in single component filtration of BSA and lysozyme.

be positively charged at all these pH values. It would therefore form a positively charged, self-rejecting layer on top of the polysulfone membrane. This would result in low transmission of positively charged lysozyme molecules through the membrane due to long range electrostatic repulsion effect as shown in Fig. 3. As the pH is increased, the magnitude of positive charge on lysozyme would decrease, resulting in lower repulsion effect and hence higher transmission of lysozyme. The highest transmission can be expected at pH 11.0, that being the pl of lysozyme. The pl of BSA is 4.9 and so it is expected to be negatively charged at all the pH values examined. It will therefore experience repulsion at the membrane surface at these pH values due to the intrinsic charge on the polysulfone membrane surface. The repulsion effect is expected to be minimum at lower pH values as magnitude of negative charge on both membrane, and BSA would be lower. This would probably explain the higher observed transmission at lower pH. The highest transmission of BSA can be expected at pH 4.9 (its isoelectric point),

With lysozyme, the flux is not so strongly dependent on pH. A slightly higher flux value at lower pH is due to lower accumulation of proteins on the merebrane surface as a result of the electrostatic repulsion effect. The flux is, however, very much pH dependent with BSA. Higher flux values are observed at higher pH due to lower accumulation of proteins on the membrane surface due to greater repulsion effect. With a decrease in pH, concentration polarization will be greater, due to lower repulsion effect from the membrane surface.

3.2. Fractionation of lysozyme and BSA using native membrane Effect of pH on the fractionation of BSA and lysozyme when filtering a binary mixture was examined at five different pH values, i.e. 5.2, 6.0, 7.0, 8.0 and 8.8. The concentrations of BSA and lysozyme in the feed were 0.5 kg m -3 each. The permeate flux, solute transmission and selectivity values obtained at different pH are shown in Table 1. At pH 5.2, ~'~'= 3.3

R. Ghosh, ZF. Cui/Journal of Membrane Science 139 (1998) 17-28

22

Table 1 Fractionation of BSA and lysozyme at different pH pH

5.2 6.0 7.0 8.0 8.8

Permeate flux (kgm-es-lxl0 3.44 3.29 3.27 3.06 3.32

Tob for BSA 3) 25.70% 4.00% 0.59% 0.50% 0.45%

Ji for BSA

Tob for lysozyme

Ji for lysozyme

( m m o l m - 2 s i) 6.598x 10-6 9.821 x 10 -6 0.144x 10 -6 0.114x 10 -6 0.111 x 10 -6

85.40% 96.10% 98.90% 98.10% 98.98%

1.041 )< 10 4 1.121 × 10 4 1.146× 10 4 1.064x 10 4 1.165 × 10 4

~

.~

3.3 24.0 167.6 196.2 220.0

15.8 114.1 795.8 933.3 1049.6

(mmolm 2s-1)

Membrane: PS 50 kDa MWCO. Feed: 0.5 kg m 3 BSA+0.5 kg m 3 lysozyme. Buffer: 20 mM phosphate at different pH. TMP: 20 kPa. Stirrer speed: 1400 rpm.

force

repulsive force

Potysulfonemembrane

lysozymelayer

L ~+= L ~ ]

Fig. 3. Electrostatic interaction between the polysulfone membrane and the lysozyme molecules.

while at pH 8.8, ~ = 220. Similarly, the molar selectivity (~M) ranged from 15.8 at pH 5.2 to 1050 at pH 8.8. The physical meaning of a ~M value of 1050 is the transmission of that number of lysozyme molecules through the membrane per molecule of BSA. From a comparison of Fig. 2 and Table 1, one can immediately see that the transmission of lysozyme is consistently higher in the filtration of a binary mixture at all pH values, in comparison to single component filtration. In contrast, the transmission of BSA is higher in binary filtration only at pH 5.2. The

increased transmission of lysozyme can be explained in terms of the presence of negatively charged BSA molecules in the feed. The accumulation of BSA molecules near the membrane surface presumably reduced the extent of formation of the positively charged layer by lysozyme molecules. Hence, there is greater transport of lysozyme through the membrane. On the other hand, the decrease in BSA transmission could be due to heteroaggregation of lysozyme and BSA, which are oppositely charged at all the pH values examined. The heteroaggregation

R. Ghosh, ZF. Cui/Journal of Membrane Science 139 ~1998j 17-28

of BSA and lysozyme near the pore is not likely to affect the transmission of lysozyme significantly due to the high transmission of lysozyme relative to that of BSA. This is based on the assumption that such electrostatic heteroaggregation would involve equimolar participation of BSA and lysozyme. It was observed that permeate flux in BSA/lysozyme binary ultrafiltration was greater than in single component ultrafiltration of BSA. This was consistently observed and the reason for this is under investigation, Ultrafiltration experiment was carried out for 80 min in order to see if the fractionation can be carried out for extended periods of time. The feed comprised 0.5 kg m 3 each of BSA and lysozyme in 20 mM sodium phosphate buffer and the operating pH was set to 8.8, at which highest selectivity was observed before. The flux and transmission data obtained are shown in Fig. 4. The flux and transmission values are more or less consistently stable over the entire duration of the filtration run. This would indicate that the fractionation process can be run for

23

longer durations without significant loss of flux or selectivity. Thus, by manipulating the pH of the feed solution, the molar selectivity of separation of BSA and lysozyme can be increased tYom 15.8 at pH 5.2 to 1050 at pH 8.8. Fractionation studies were also carried out at higher feed concentration (2 kg m -3 each of BSA and lysozyme) at pH 8.8. The flux, transmission and selectivity data are shown in Table 2. The transmission of BSA is similar to that observed at lower feed concentration but there is drastic reduction in the transmission of lysozyme from 99% to about 61%. A molar selectivity (ti'M) value of 532 is observed, almost half of that observed at low feed concentration. This lower selectivity may be due to the formation of more intense self-rejecting adsorbed lysozyme layer at higher lysozyme concentration. Selectivity is often a rather misleading parameter, as it is more sensitive to changes in the value of the denominator (=oh 2), particularly when its value is less than 1%. Hence, selectivity alone may not be adequate in describing the efficiency of a separation process.

100

4.00E-03

3.50E-03 8O

n

O

•~

[]

D..-------'O---..~

.3.00E-03 ~'e

6o.

E

~'"" •l~ •

• 2.50E-03 Membrane: PS 50 kDa Feed: 0.5 kg m "3 BSA

40

+ 0.5 kg m "3 lysozyme.

.~

O

~

•

T r a n s m i s s i o n (lysozyme'.

•

Transmission (BSA) Permeate

flux

Buffer: 20 mM phosphate, pH 8.8 Stirrer speed: 1400 rprn 20 1.50E-03

0

: -" 0

500

:, 1000

~

; 1500

-"

" 2000

~ ', 2500

~' 3000

', -' 3500

:

-=

4000

Time (sec.) Fig. 4. Variation of permeate flux and transmission with time.

: 4500

-'

=

¢= ¢~ 2,00E-03

TMP: 20 kPa

X

1.00E-03 5000

I~

24

R. Ghosh, ZF. Cui/Journal of Membrane Science 139 (1998) 17-28

Table 2 Fractionation of BSA and lysozyme using 5 0 k D a native polysulfone membrane

Pure water flux (kgm-2s -1) Flux (kg m -2 s -1)

0.004950 0.002065

%b for BSA %b for lysozyme Selectivity 0h) Solute flux for BSA (mmol m -2 s-1) Solute flux for lysozyme (mmol m 2 s-~)

0.55% 61.63%

Molar selectivity ( ~ )

532

112

3.3903 x 10 7 1.8052x 10 4

Membrane: PS 50 kDa MWCO. Feed: 2 kg m -3 BSA+2 kg m -3 lysozyme. Buffer: 20 mM phosphate, pH 8.8.

TMP: 20 kPa. Stirrer speed: 1400rpm. For example, a process where the ~-obvalues of the two solutes are 100% and 1%, respectively, is more effcient in terms of throughput of product than one where the values are 10% and 0.1%, respectively, even though they have the same selectivity. For a separation process to be attractive, both selectivity and transmission of the solute desirable in the permeate should be high. There is hence a need to enhance the transmission of lysozyme, while at the same time keeping the selectivity high in order to make the process more efficient and acceptable.

5 min. All permeate and retentate samples obtained during ultrafiltration experiments with pretreated membranes were analysed by an FPLC analytical protocol. Myoglobin was absent in all permeate as well as retentate samples. This clearly indicates that all loosely bound myoglobin is removed during the rinsing operation and the first 5 rain of the compaction process. The myoglobin molecules bound to the membrane are irreversibly adsorbed (under operating c o n ditions) and are hence not removed during fltration operation.

3.3.2. Myoglobin loading The amount of myoglobin adsorbed on the merebrane was quantified at different operating pH using material balance. This is not a very accurate technique but nevertheless gives an approximate idea about the extent of adsorption. Maximum adsorption was observed at pH 7.0 as shown in Fig. 5. The amount of myoglobin adsorbed on polysulfone membrane at pH 7.0 is about 7 x 10 - 7 k g m - 2 . This indicates that adsorption of myoglobin on polysulfone is not due to electrostatic interactions but due to other types of interactions such as hydrophobic interactions, van der Waals forces, etc., since at pH 7.0, myoglobin is uncharged. 1.2

3.3. Membrane pretreatment to enhance lysozyme transmission Ultrafiltration experiment were carried out with membranes pretreated by passive adsorption of myoglobin at the same pH as in previous experiments, i.e. 5.2, 6.0, 7.0 8.0 and 8.8.

~ i

1

~ 0.8 ~

3.3.1. Stability of pretreatment

~ 0.6

The stability of any pretreatmentadsorption method is critical profor its successful application. The of tein on hydrophobic membrane surface is normally expected to be irreversible under normal operating conditions. Thus, the likelihood of the adsorbed myoglobin being displaced by lysozyme during ultrafiltration is minimal. This is confirmed in all the experiments. The filtrate samples obtained during membrane compaction after pretreatment were analysed for the presence of myoglobin. No myoglobin could be detected in filtrate samples obtained after

~ 0.4 i

~

~

E ~ o.2

0

~ 5.2

~ 6

, 7

8

8.8

pN Fig. 5. Myoglobin

adsorptionon

polysulfone

membrane.

R. Ghosh, Z E Cui/Journal of Membrane Science 139 (1998) 17-28

25

0.006,

120

Y

o

"

1oo • rn~mbrane)

'w ~'E

/ / ~

=~ 0.004

~

Flux (Pret reated membrane)

~

Transmission (Native membrane)

E

==

-

-4o

Membrane: PS (native and pretreated) Feed: 0.5 kg m"~ lysoz'/me TMP: 20 kPa Buffer: 20 mM phosphate Stirrer speed: 1400 rpm

0.002 5

¢

._o w

~

• a.

0.003

80

I 6

lID ,Q

O 20

I 7

I 8

0

pH

Fig. 6. Lysozyme uhrafiltration with native and pretreated polysulfone membranes.

3.3.3. Effect on waterflux Pure water flux was determined for the native and the pretreated membranes. The results indicate that the hydraulic permeability of the membrane pretreated by passive adsorption was about 8% lower than that of the native membrane which indicates that adsorption of myoglobin results in some pore blocking,

3.3.4. Lysozyme ultrafiltration using pretreated membranes Each run was carried out for 20 min, at 20 kPa transmembrane pressure using 0.5 kg m -3 lysozyme in appropriate buffer as feed solution. The transmission and flux data obtained from these experiments are shown in Fig. 6. Results obtained from protein ultrafiltration experiments indicate that with membranes pretreated by passive adsorption of myoglobin, the permeate flux values are slightly higher than those observed with native membrane. This is due to the fact that pre-adsorption of myoglobin prevents accumulation of lysozyme molecules by electrostatic interaction, close to the membrane surface, thus resulting in greater permeate flux. The results clearly show that there is pronounced enhancement of transmission of lysozyme with respect to native membrane at all pH values. At and above pH 6.0, observed transmission of lysozyme is

just above 100% and this value does not change very appreciably with pH. The mechanism of transmission enhancement can be explained in terms of the prevention of formation of a positively charged, sellrejecting lysozyme layer on top of the polysulfone membrane during ultrafiltration. Pretreatment with myoglobin would ensure the formation of a myoglobin layer on top of the hydrophobic polysulfone membrane. This would prevent long range electrostatic interactions between the positively charged lysozyme molecules and the negatively charged membrane, thus preventing the formation of the positively charged lysozyme layer, which repels lysozyme molecules from approaching the pores. The maximum transmission of lysozyme through pretreated membrane is observed at pH 7.0, when myoglobin has no net charge. At this pH, adsorbed myoglobin can act as an uncharged, shielding layer which is very effective in preventing the formation of self-rejecting lysozyme layer. At pH values lower than 7.0. myoglobin is mildly positively charged. This also prevents the formation of the self-rejecting lysozyme layer, butdue to slight repulsion effect between adsorbed myoglobin layer and the lysozyme in solution, the transmission of lysozyme is lower than that observed at pH 7.0. At pH values higher than 7.0, myoglobin has a mild negative charge and there is the likelihood of formation of a

26

R. Ghosh, ZF. Cui/Journal of Membrane Science 139 (1998) 17-28

secondary layer of lysozyme on top of the myoglobin layer. However, from experimental data it is evident that the self-rejecting property of such a secondary layer is not significant when compared with that of the primary layer of lysozyme formed on a native merebrahe. Hence, even at pH values higher than 7.0, there is enhancement in lysozyme transmission due to pretreatment. The transmission enhancement effect is more evident at lower pH when the lysozyme molecules carry a higher charge, Another reason for highest observed transmission of lysozyme through pretreated membrane at pH 7.0 is the maximum adsorption of myoglobin at this pH. This results in better shielding of lysozyme molecules from the membrane surface. The long range electrostatic interactions between proteins in solution and the membrane surface, are described by the DLVO (Derjaguin, Landau, Verwey and Overbeek) theory [19]. Beyond a certain distance from the surface known as the Debye length, interactive forces are absent. When proteins adsorb on membrane surfaces, the monolayer spans the whole of the Debye length [19]. Thus, it can be assumed that adsorption of myoglobin effectively prevents any form of long range electrostatic interaction between the charged membrane surface and the charged lysozyme molecules, The surface available to lysozyme molecules during ultrafiltration is altered by pretreatment. The characteristics, in particular the surface charge are different from the native polysulfone membrane. Ehsani et al. [18] have shown that charge on a membrane surface is altered by myoglobin adsorption. Using streaming potential measurements, they found that at pH 5.0, polysulfone membrane was shown to have a zeta potential of - 3 . 0 mV. Pretreatment by adsorption of 10, 20 and 40 ppm myoglobin changed the surface zeta potential values to -5.2, - 3 . 7 and -0.5, respectively. Thus a higher concentration of myoglobin was found to be more effective in lowering the charge on the membrane surface, as more myoglobin is adsorbed on to the membrane surface. In our experiment, polysulfone membrane was treated with 0.5 kg m -3 myoglobin solution. The amount of myoglobin adsorbed is expected to be higher and hence the effect is more significant, The observed greater than 100% transmission of lysozyme indicates that there is some kind of associa-

tion effect between the adsorbed myoglobin layer and the solute (lysozyme). Since the interaction between myoglobin and the polysulfone membrane is hydrophobic in nature, the hydrophobic residues of myoglobin are likely to orient towards the membrane surface resulting in the hydrophilic residues being orientated the aqueous media. Thus, the pretreated membrane surface will be much more hydrophilic than the surface of the native membrane resulting in higher transmission of lysozyme.

3.4. Fractionation of BSA-lysozyme mixture using pretreated membrane The effect of membrane surface pretreatment on fractionation of BSA and lysozyme was investigated at pH 7.0 and 8.8 using a feed concentration of 2 kg m 3 each of BSA and lysozyme, pH 7.0 and 8.8 were selected as the transmission of lysozyme through the pretreated membrane was found to be highest at pH 7.0, while the transmission through native membrane was found to be maximum at pH 8.8. The flux, transmission and selectivity data are summarized in Table 3. From the results, it is evident that the membrane pretreatment can improve protein fractionation. There is near complete rejection of BSA and very high transmission of lysozyme. At pH 7.0, with the native membrane 328 molecules of lysozyme pass through the membrane per molecule of BSA. At the same pH, with the pretreated membrane the figure is about 646 molecules of lysozyme per molecule of BSA. Thus, the molar selectivity is almost doubled with the pretreated membrane. At pH 8.8, transmission of lysozyme through pretreated membrane is considerably lower than that observed with pretreated membrane at pH 7.0. However, the selectivity is not that significantly lower, primarily due to lower transmission of BSA at that pH. It is also found that at pH 7.0, myoglobin coating can also enhance permeate flux, although the pure water flux is 25% higher with the native membrane. The permeate flux is higher (~11%) with the pretreated membrane. This is due to the fact that the preadsorption of myoglobin on the membrane reduces the interactions between the proteins (BSA and lysozyme) and the membrane, and results in much lower adsorption of protein during ultrafiltration. This outweighed

R. Ghosh, ZF. Cui/Journal o['Membrane Science 139 (1998)17-28

27

Table 3 Fractionation of BSA and lysozyme using pretreated membrane Parameters

Native membrane

Pretreated membrane

pH 7.0

pH 7.0

pH 8.8

Pure water flux (kg m 2s 1) Permeate flux in protein experiment (kg m 2 s l) T,,b for BSA r,,b for lysozyme Selectivity (~,') Ji for B S A ( m m o l m 2s i) J~ for lysozyme (mmol m 2 s i) Molar selectivity (~"M)

0.00506 0.00254 0.93% 67.76% 75.82 7.07x10 7 2.276× 10 a 328

0.00381 0.00281 (/.67% 90.41% 139.89 5.63×10 7 3.500× 10 4 646

().00396 ().()0209 0.54c/( 71.3% 132.1)3 3.374×1II v 2.112× 10 4 626

Feed: 2 kg m 3 BSA+2 kg m 3 lysozyme. Buffer: 20 mM phosphate. TMP: 20 kPa. Stirrer speed: 1400 rpm.

the pore blocking effect of the adsorbed myoglobin and hence resulted in higher permeate flux in protein ultrafiltration, The lower transmission of BSA through the pretreated membrane can also be attributed to partial pore blocking by myoglobin. The dimension of BSA is comparable to that of the pore while lysozyme is smaller. Hence, partial pore blocking can significantly reduce the transmission of BSA. However, the blocking is not extensive enough to affect the transmission of lysozyme or significantly lower the hydraulic permeability, At pH values greater than 7.0, there should theoretically be repulsion of BSA molecules away from the pretreated membrane surface since both myoglobin and BSA possess net negative charge. However, Tables 2 and 3 show that reduction in BSA transmission through pretreated membrane is more extensive at pH 7.0 as compared to that at pH 8.8. Hence, it may be concluded that electrostatic interaction effect is more significant in the case of lysozyme and less significant in the case of BSA. Lysozyme is a small, compact molecule and is more likely to behave like a rigid particle with clearly defined charge. On the other hand, BSA being a large flexible molecule is less likely to behave similarly. From a comparison of Tables 2 and 3, it is evident that efficiency of fractionation at higher feed concentration is better when filtering BSA/lysozyme mixture through pretreated membrane at pH 7.0 than when filtering the mixture through either pretreated or native

membrane at pH 8.8. Thus, a combination of pH effect and membrane pretreatment can be used for efficiently fractionating BSA and lysozyme.

4. Conclusions 1. The transmission behaviour of lysozyme and BSA in binary ultrafiltration (using 50 kDa MWCO polysulfone membrane) is different from that observed in single protein ultrafiltration. At all the pH values examined, consistently higher transmission is observed with lysozyme in binary ultrafiltration. On the other hand, the transmission of BSA was generally lower in binary ultrafiltration. This phenomenon works in favour of fractionation of BSA and lysozyme. 2. Separation of BSA and lysozyme using a 50 kDa MWCO polysulfone membrane is very strongly dependent on pH. At pH 8.8. and low teed concentration, a very high selectivity value (.~;,) of 220 was observed for the BSA/lysozyme system. Studies carried out for extended durations showed that neither flux nor selectivity declined significantly during the ultrafiltration operation. 3, Surface pretreatment by myoglobin adsorption is effective for enhancing the transmission of lysozyme through a 50 kDa MWCO polysulfone ultrafiltration membrane. An increase in transmission between 20-60% was observed, being dependent on the operating pH.

28

R. Ghosh, Z.F. Cui/Journal of Membrane Science 139 (1998) 17-28

4. Membrane pretreatment can also enhance the permeate flux. 5. Membrane pretreatment can aid the fractionation of BSA and lysozyme even at higher feed concentrations. 5. List of symbols J Sa %b Cb

Permeate flux (kg m -2 s - l ) Apparent sieving coefficient (dimensionless) Observed transmission of a solute (dimensionless) Bulk concentration of solute (kg m - 3 )

Cp ~b Ji ~

Permeate concentration of solute (kg m -3) Selectivity of solute separation (dimensionless) S o l u t e f l u x ( m M m - 2 s 1)

Molar selectivity (dimensionless)

Acknowledgements T h i s w o r k is supported by the Biotechnology and Biological S c i e n c e R e s e a r c h C o u n c i l (BBSRC) [GR

SPC 05236]. R. Ghosh is grateful to the Association of Commonwealth Universities for awarding a Commonwealth Scholarship for conducting studies at the University of Oxford. The authors would like to thank

(IgG) by selective filtration, Biotechnol. Bioeng. 43 (1994) 960.

[51 R.G. Nel, S.F. Oppenheim, V.G.J. Rodgers, Effect of solution

properties on solute and permeate flux in bovine serum albumin - IgG ultrafiltration, Biotechnol. Prog. 10 (1994) 539. [61 S. Lentsch, R Aimar, J.L. Orozco, Separation of albuminPEG: Transmission of PEG through ultrafiltration membranes, Biotechnol. Bioeng. 41 (1993) 1039. [7] L. Zhang, H.G. Spencer, Selective separation of proteins by microfiltration with formed-in-place membranes, Desalination 90 (1993) 137, [8] N.N. Sudareva, O.I. Kurenbin, B.G. Belenkii, Increase in efficiency of membrane fractionation, J. Membr. Sci. 68

(1992) 263. [9] K.C. Ingham, T.F. Busby, Y. Sahlestrom, F. Castino, Separation of macromolecules by ultrafiltration: Influence of protein adsorption, protein-protein interactions, and concentration polarization, in: A.R. Cooper (Ed.), Polymer Science and Technology, Vol. 13 (Ultrafiltration Membranes and Applications), Plenum Press, New York, 1980, p. 141. [10] S. Nakatsuka, A.S. Michaels, Transport and separation of proteins by ultrafiltration through sorptive and non-sorptive membranes, J. Membr. Sci. 69 (1992) 189. [11] L. Millesime, J. Dulieu, B. Chaufer, Fractionation of proteins using modified membranes, Bioseparation 6 (1996) 135. [12] S. Nakao, H. Osada, H. Knrata, T. Tsuru, S. Kimura, Separation of proteins by charged ultrafiltration membranes, Desalination 70 (1988) 191. [13l A. Higuchi, S. Mishima, T. Nakagawa, Separation of proteins by surface modified polysulfone membranes, J. Membr. Sci. 57 (1991) 175.

[14] L. Millesime, C. Amiel, B. Chaufer, Ultrafiltration of lysozyme and bovine serum albumin with polysulfone membranes modified with quaternized polyvinylimidazole,

Prof. R.W. Bowen, University of Wales Swansea, for his useful comments and suggestions on this work. The authors would also like to thank the referees for their valuable comments and suggestions,

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