- Email: [email protected]
Membrane application in proteomic studies: Preliminary studies on the effect of pH, ionic strength and pressure on protein fractionation
DESALINATION Desalination 179 (2005) 381-390
ELSEVIER
www.elsevier.com/locate/desal
Membrane application in proteomic studies: Preliminary studies on the effect of pH, ionic strength and pressure on protein fractionation M.M.D. Zulkali*, A.L. Ahmad, C.J.C. Derek School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal, Seberang Perai Selatan, Pulau Pinang, Malaysia Tel. +60 (4) 593-7788, ext. 6414; Fax." +60 (4) 594-1013; email: [email protected]
Received 29 September 2004; accepted 22 November 2004
Abstract
The effects ofpH, ionic strength and pressure on membrane-modulated protein fractionation were studied. Model protein, bovine serum albumin (BSA), was subjected to various pH, ionic strengths and pressures. The protein content and salt rejection in the retentate and flux were monitored. The design of the experiment was carried out with the aid of response surface methodology (RSM). A set of 20 designed experiments was carried out, and the data collected were analyzed using central composite experimental design in response surface methodology (RSM) by Design Expert, Version 5.0.7 (StatEase, USA). The response surface plot for fluxes was higher at lower ionic strengths and at high pH due to the shielding of charge and the electrostatic repulsion of protein with a similar charge while higher pressure reduced the flux through concentration polarization and expansion of adsorbed BSA on the membrane surface. Chloride rejection was highest at pH 8 and was independent of the salt concentration where the negative charge of BSA was stronger. It was observed that higher pressure seemed to favor rejection. Keywords: Ionic strength; Flux; pH; Response surface methodology
1. Introduction
Purification o f protein is often carried out using the chromatographic method, which is very specific, and a very pure fraction o f protein can *Corresponding author.
be obtained. However, the method is difficult to scale up, yielding a low throughput with an extremely high cost. Therefore, membranes are seen as an alternative that is cost effective and can be fine-tuned to achieve high productivity and purity at the same time [1-4]. Ultrafiltration
Presented at the conference on Membranes in Drinking and Industrial Water Production, L 'Aquila, Italy, 15-17 November 2004. Organized by the European Desalination Society.
0011-9164/05/$- See front matter © 2005 Elsevier B.V. All rights reserved doi: 10.1016/j.desal.2004.11.084
382
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
(UF) membranes can be used to separate protein using the same principle as chromatography as well as its natural separation, which is size dependent. If only the sizes of the pores are used as the means of separation with membranes, mostly 10-fold differences in molar masses of the proteins are needed because of the dispersed membrane pore sizes and fouling of the membranes. It is generally accepted that solutes whose molecular dimensions are small enough to permeate the membrane are substantially retained by the same membrane when larger solutes are present [5,6]. However, studies have shown that membrane filtration of proteins can also be drastically influenced by the nature of a solutesolute interaction [5-8]. These physicochemical interactions can and do occur between the membrane and solutes in the form of electrostatic charge, hydrophobic or even charge transfer. As a result, transmission of solutes through the membrane can either be manipulated by the physicochemical parameters such as pH and ionic strength or the operational parameters such as transmembrane pressure, system configuration, etc. The solute-solute interaction will determine the filtration rate and the rejection of solute when one or more proteins are ultrafiltered. Pujar and Zydney [9] showed that the rejection coefficient of bovine serum albumin (BSA) could be increased from about 0.2 to more than 0.995, simply by lowering the ionic strength. The dramatic increase in protein rejection is due to the strong electrostatic repulsion of the negatively charged protein from the pores of membrane. It is usually observed that proteins have the highest transmission at their isoelectric point (IEP) and lower transmissions when not at their IEP values [5,6,10,11]. For charged molecules such as proteinS, transport of rejected material back from the membrane surface to the bulk solution is not only dependent on the concentration gradient at the membrane interface but is
also driven by the electrostatic repulsion between the charged molecules. Owing to their charge repulsion, the protein will not be easily transmitted through a pore close to the size of the protein with a similar charge. It is mostly transmitted at its IEP where it has no charge. The effect of salt on filtration of protein can be seen as shielding of charges. Charged proteins, which normally do not penetrate the membrane, gain transmission through the membrane due to the shielding of charges. This shielding effect reduces the hydrodynamic diameter of a protein differing from its IEP. The shielding effect also naturally decreases the charge of the membrane. However, when proteins are at their IEP, normally the opposite effect is observed. The presence of salt decreases the retention of charged proteins and mostly increases the retention of neutral proteins. These effects have been shown by Fane and coworkers [8,12] and Nystrom et al. [13]. The hydrodynamic radius of the protein can thus be manipulated using salt. Pujar and Zydney [14] showed the influence of solution ionic strength when it was reduced from 150 to 1.5 mM and subsequently decreased the protein transmission by more than two orders of magnitude. The best fractionation was achieved when one of the proteins was at its IEP and the other one was retained due to the charge repulsion with the membrane as an example myoglobin and BSA at pH 4.8 [13]. The effect of the pH and salt concentration can be very profound. It is not limited to UF membranes and is found also in microfiltation (MF) membranes. As an example, BSA transmission can be as low as 40% when a MF membrane with 0.16 #m pores, which is definitely much larger than the molecule itself, is used [15]. In order to exploit effectively the electrostatic interaction for protein separation, it is important to evaluate the magnitude of these effects accurately. Noordman et al. [5] have shown that rejection and fluxes of BSA retention can be well
383
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
described by a model based on the MaxwellStefan theory of mass transfer with the nonidealities through the Carnahan-Starling equation of state for rigid spheres. The study was carried out by obtaining data for a single protein over a wide range of different environmental conditions, similar to those reported in literature elsewhere with respected to electrostatic interactions [7,9]. In the present study, an attempt was made to access the magnitude of pH, ionic strength and pressure on the protein and salt rejection by UF with the aid of response surface methodology (RSM).
valve
7 ~ 2 ~ Amicon
0- © Sd u b on reservoir
i Fig. 1. Ultrafiltration set-up. 0.45 0.4 0.35 ~"
2. Material and methods
2.1. Materials Protein solutions of appropriate concentrations were prepared. The model protein was BSA (Fraction V, Sigma Aldrich). BSA has a tertiary structure with an IEP of 4.9 and a molecular weight of 67,000 Da [16].The pH was adjusted with NaOH (Fluka, BioChemika) or HC1 (Fluka, BioChemika) and the ionic strength was adjusted via addition of solid crystals of NaC1 (Fluka, BioChemika). The protein solution was prepared no longer than 1 h before use. All solutions were prepared using deionized water. 2.2. Equipment and measurement The experiment was carried out in a feedbatch configuration as shown in Fig. 1. A circular flat-sheet membrane was placed on the bottom of an Amicon 8200 stirred cell supported by a porous plate. The effective area of the membrane was 28.27 cm 2. The cell contained a single blade stirrer driven by magnetic coupling at 500 rpm. The membrane cell was pressurized with nitrogen gas to the solution reservoir at the required pressure and that at the permeate side was at atmospheric. All experiments were carried out
0.3
~E g.. 0.25 g 0.2 o.15 IJ-
0.1 0.05 "-r--
1
2
3
4
Pressure (bar)
Fig. 2. Hydraulic permeability of polyethersulfone UF membrane (30 kDa). under ambient temperature. Mixing of the feed solution was accomplished by the stirring of the solution. An UF polyethersulfone flat-sheet membrane (PBTK), acquired from Millipore, with a 30,000 kDa molecular weight cut-off was used. The membranes were rinsed in deionized water for 1 h before use, and pretreatment of the membrane was carried out at 450 kPa for 1 h. The hydraulic permeability of the membrane was then determined (Fig. 2). Solution of 1 g/L of BSA was filled into the Amicon 8200 stirred cell. The loss of volume in the cell due to filtration was balanced with the similar concentration of BSA from the reservoir. The membrane was cleaned after each run. First, the membrane was rinsed with deionized water and then treated with 0.1 M NaOH solution for
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30 rain at the same pressure as that used in the subsequent experiment. Then the membrane was flushed thoroughly with deionized water. The pure water flux was measured after the cleaning to evaluate the performance of the cleaning step. The flux was maintained within 5% difference from the original pure water flux. The permeate fluxes were measured gravimetrically. Concentrations of BSA both in permeate and concentrate were determined by UV absorption at 280 nm. The concentration of sodium chloride was determined through mercuric titration (chloride test kit, Hanna Instrument). The rejection of BSA turned out to be always larger that 99%, and therefore it was assumed to have 100% rejection in all the experiments.
2.3. Experimental design RSM is a useful technique in designing experiments for studying the effect of several factors influencing responses by varying them simultaneously and carrying out a limited number of experiments with the objective to optimize the response as oppose to the conventional method of optimization that involves varying one parameter at a time while keeping the others constant. RSM has been applied to optimize numerous biotechnological processes [17-19] due to its ability to include the interactions of several factors (or variables) that affect the response of a particular process. A central composite rotatable design (CCRD) was used to obtain a better understanding of the relationship between the variables of pH and ionic strength toward the response of flux, protein and ion retention. A 23 full factorial CCRD with five levels was used in the study which required 20 set of experiments. The variables and levels selected for the study on protein rejection were pH (3.5-8), NaC1 concentration (1.3-5 g/L) and pressure (2-4 bar). The range and the levels of the variables investigated in the research are given in Table 1. Table 2 shows the
actual experiment carried out for developing the model. The experimental data were analyzed by the response surface regression procedure to fit a second-order polynomial equation: k
k
Y = DO+ Z ~iXi + Z ~iiX? + Z Z i:1 i+1 i
~ijXiXj (1)
where y is the response, 13i the constant coefficient, and x~ the independent variable. The regression analysis, statistical significance and graphical analysis were done using Design Expert (Version 6.0, StatEase, USA) software. The statistical significance of the regression coefficients was determined by the Student t-test, the second-order model equation was determined by the Fischer's test and the proportion of variance explained by the model obtained was given by the multiple coefficient of determination, R 2.
3. Results and discussion
The effects of ionic strength, pH and pressure on flux and chloride rejection were studied using central composite design in RSM. The data analysis was preformed on a total of 20 sets of experiments consisting of eight factorial points, six axial points and six center points. The range chosen for the process variable were 3.5-8 for pH, 1.3-5 g/1 of NaC1 for ionic strength and 24 bar for pressure. A regression analysis was preformed to fit both the response function of flux and chloride rejection. Two equations were obtained through the program for each of the response. The model expressed by Eqs. (2) and (3), where the variables take their coded values, represents the percentage of chloride rejection (Ii,.) and flux (Yi) as a function of pH (x 0, ionic strength (x2) and pressure (x3). The coefficient of the second-order polynomial equation is given in Eqs. (2) and (3).
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
385
Table 1 Experimental domain Variables
Coded/range and level
x~ - - pH x2 - - NaC1 (g/L) x3- - Pressure (bar)
-2
-1
0
+1
+2
1.96 0.04 1.32
3.50 1.30 2.00
5.75 3.15 3.00
8.00 5.00 4.00
9.53 6.26 4.68
Table 2 Fxperimental data for RSM-CCRD analysis Run
xI
x2
x3
Flux (m3/mZ,h)
Chloride rejection (%)
1 2 3 4
0 0 0 0 - 1 0 0 -1 +1 -2 -1 0 +1 +1 0 +1 +2 0 0 -1
0 0 0 +2 +1 0 0 -1 -1 0 -1 0 +1 +1 0 -1 0 0 -2 +1
0 0 0 0 -1 0 0 +1 -1 0 -1 0 -t +1 -2 +1 0 +2 0 +1
0.129 0.136 0.101 0.133 0.145 0.132 0.197 0.081 0.178 0.131 0.168 0.147 0.135 0.139 0.140 0.144 0.127 0.156 0.140 0.080
3,19 1.56 9.09 t5.79 3.50 -3.79 7.69 -3.05 7.37 5.66 5.69 5.26 12.38 30.86 -4.20 3.18 13.55 3.82 -2.50 12.06
5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Yr = 3.72 + 3.58x 1 + 5.59x1 + 2.02x 3 + 2.79x 2 + 1 . 7 4 x ~ - 0.67x 2 + 2 . 4 7 x l x 2 + 1.81XlX 3 + 5.00xzx 3(2)
Y f : 0.14 + 5.679 × 10°3xl - 6.774 × 10-3x2 - 0.036x 3 + 0.015xax 2 + 0.015x 3
The fitness o f the model was expressed b y the coefficient o f determination, R 2. The R 2 for Eqs. (2) and (3) were 0.8665 and 0.5063, respectively. The R 2 value o f 0.8665 (86.65%) indicated the variability in the response o f the model where
(3)
13.35% o f the total variation was not explained b y the model [Eq. (2)], which m a y seem to be fairly good. However, Eq. (3) with an R 2 o f 0.5063 could not be explained variation by the model. The low coefficient o f determination
386
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
Table 3 ANOVA for model regression Source Flux model: Model residual Lack of fit Pure error Chloride rejection model: Model residual Lack of fit Pure error
Sum of squares
Degree of freedom
Mean square
F value
Probability level
1083.02 168.35 60.10 108.25
9 10 5 5
121.45 16.84 12.02 21.65
7.21
0.0024
0.56
0.7330
6.586 E-3 6.422 E-3 1.408 E-3 5.014 E-3
5 14 9 5
1.317 E-3 4.587 E-4 1.465 E-4 1.003 E-3
2.87
0.0547
0.16
0.9917
could be due to the constant changes of protein concentration along with the physicochemical properties as it is being concentrated, which influences the flux. Adding protein concentration as another variable may help to give a better model for prediction [22]. The ANOVA for both flux and salt rejection model is shown in Table 3. The rejection of salt observed during the protein purification is shown in the threedimensional presentation (correlation between pH, ionic strength and pressure on the chloride rejection) in Fig. 3a-c. It was observed that chloride rejection was very much dependent on the interaction ofpH, pressure and ionic strength. The chloride rejection was highest ranging from 3.73% to 28.05% at pH 8 (i.e., above its IEP), independent of salt concentration. It seems that the charge of the protein was the same as that of the chloride ions. Due to the concentration polarization and adsorption of the protein on the membrane [21], there was an accumulation of negative charges at the membrane that results in repulsion of chloride ions and attraction of the positive sodium ions. The net negative electrical potential on the membrane surface repelled the negatively charged chloride ions and thus increased its rejection. The rejection increased (with the highest increment of 18.01% rejection at pH 8, ionic strength at 5.00 g/L from a pressure
change of 2 bar to 4 bar), along with the operating pressure, partly due to the higher formation of concentration polarization at the membrane interface. On the other hand, at low pH (i.e., below the IEP) the chloride rejection was lower compared to rejection of the ion above the BSA's IEP due to the positively acquired charge of the adsorbed protein. The chloride ion, which was the counterion, then was attracted by the protein at the membrane interface. The chloride ion rejection was lowered. However, at lower operating pressures, the chloride rejection increased with lower ionic strength due to the reduction in charge shielding of the positive ion. The higher the ionic strength, the better the rejection of chloride, which was independent ofpH and pressure. The presence of a higher concentration of salts provided a better shielding of charge and thus reduced the retention effect on the membrane and concentration polarization. The effects ofpH, ionic strength and pressure towards flux are shown in Fig. 4a-c. It was seen that the fluxes behaved as expected from the electrostatic consideration point of view. The flux increased with decreasing ionic strength due to lower shielding of protein charges at low salt concentration. Therefore, the protein molecules strongly repelled each other, especially at the
387
M.M.D. Zulkali et al, / Desalination 179 (2005) 381-390
(a)
(b) 19.89 28,05
14.86
19.90
9.83
11.75
8 "~
3.6~
,~
4,7S
"~
-0.2,4
-4.5
_o
5.0 O0
B:lonic
ton
1.30 3.50 1,30 3.50
Fig. 3. Response surface plots of the effect of ionic strength and pH on chloride rejection at constant pressure. (a) 4 bar, (b) 3 bar, (c) 2 bar.
(c) 10.40 7.~ 5.57 3.1~ 0.7, "m o
B:ion
membrane surface where the concentration was high. The electrostatic repulsion increased the mass transfer of protein at the membrane interface back to the bulk solution. The concentration and thus, the osmotic pressure at the membrane surface were reduced and higher fluxes were observed. The effects of solution pH on fluxes can also be in Fig. 4a-c. The fluxes were higher at higher pH (i.e., above the protein's IEP). This
phenomenon was similar to that observed in the study on the effect of ionic strength. The higher the pH, the more negatively charged the protein and their electrostatic repulsion on the membrane surface was much stronger. The concentration polarization was reduced at the membrane surface and a higher flux was observed. However, at lower pH (below the protein IEP), the fluxes decreased with increased pressure due to the formation of concentration polarization on the surface of the membrane. It was reported that below the protein's IEP, the BSA molecules expanded with decreasing pH [22]. Owing to this effect, the mobility of the molecule also decreased, which led to more profound concentration polarization on the membrane surface. Therefore, the concentration polarization was enhanced and flux deteriorated. The increased of pressure seemed to affect the flux more when the solution pH was lower due to higher extend of concentration polarization at the membrane surface compared to when the pH was higher. The curvature of the response plots in Fig. 4a-e suggest a rapid decline in the flux in
388
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
(a)
(b) 0.174
0.16~
0.156
0.14
0.13~
0,1.~
0.12~
0.1
0.1C n-
C: 2,00
2.OO 3,50
3.50
Fig. 4. Response surface plots of the effect of pH and pressure on flux at constant ionic strength. (a) 1.3 g/L,
(c)
(b) 3.15 g/L, (c) 5.00 g/L. 0.I60 0,143 0
~E
C: Pressure (bar) O
0,1~7 -
125
0.10' 008 0.167 -
@
% 0,137x LL
0t06
C:
-
= D£sir~r] Pl:li!~i;
2 O0 350
m C- 2.000 A C + 4.009
0.076 -
response to the combined effect of pH between 3.5 to 5.75 and pressure between 2.5 bar to 3.5 bar. The decline in flux could be due to the conformational change of BSA influenced by the combined effect of pressure and pH. It was postulated that the molecule expanded and affects the transmission across the membrane surface. The effect of interaction of pH and pressure towards the flux at constant ionic strength of
I 3,50
I 4,63
I 5.75
I
i
6,88
8.00
A: pH
Fig. 5. Graph plotting interaction of pH and pressure towards flux at ionic strength of 3.15 g/L.
3.15 g/L is shown in Fig. 5. The flux decreased around 45% when pH was changed from 8 to 3.5 at high pressure, but at a low pressure the
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
pressure lowered the flux by increasing the density of concentration polarization and at low pH (pH 3.5); the adsorption of protein on the membrane surface expanded and further aggravated the flux. The chloride rejection was highest at pH 8 and was independent of the salt concentration where the BSA protein had a similar charge as the chloride ion. It was also observed that higher pressure seemed to assist in the rejection.
B: I o n i c strength (g/L) 30.88
--
Desi~2.nPoints
i
B- 1.300 • 8÷ 5.000 2210
'~'
--
13.33 --
g
~ " ......
-[-
~...........
144v----....... T
0 4.57
•
~
~
389
-
• Q
-4.20 I .... :~,50
I
I
4.183
5,7~
I.... 6.88
I
References
8.00
A: pH
Fig, 6. Graph plotting interaction ofpH and ionic strength towards chloride rejection at a pressure of 3 bar. contrary was observed. It would seem that operating at high pressure and low pH was not feasible. The change of pH from an acidic to an alkaline condition seemed to have a more significant effect towards chloride rejection at high ionic strength (Fig. 6). The chloride rejection increased by more than 11% at pH 8 as compared to around 2% at low ionic strength. The results showed that the pH influence towards chloride rejection increased with the increase of ionic strength. Similar observations were made with regard to the interaction effects for other variables (Figs. 3a-c and 4a-c).
4. Conclusions It was clear that pH, ionic strength and pressure played important roles in affecting the physicochemical properties o f the protein and the transmission across UF membranes. The fluxes were higher at lower ionic strength and at higher pH (pH 8) owing to lower shielding o f charge and electrostatic repulsion o f protein molecules with similar charges. It was observed that higher
[1] R. Ghosh, S.S. Silva and Z.F. Cui, Lysozyme separation by hollow-fiber ultrafiltration, BioChem. Eng. J., 6 (2000) 19. [2] R. Ghosh and Z.F. Cui, Fractionation of BSA and lysozyme using ultrafiltration: Effect of pH and membrane pretreatment, J. Membr. Sci., 139 (1998) 17. [3] R. Ghosh and Z.F. Cui, Protein purification by ultrafiltrationwith pre-treated membrane,J. Membr. Sci., 167 (2000) 47. [4] P. Punidadas and S.S.H. Rizvi, Separation of milk proteins into fractionsrich in casein or whey proteins by cross flow filtration, Food Res. Int., 31 (1998) 265. [5] T.R. Noordman, T.H. Ketelaar, F. Donkers and J.A. Wessenlingh,Concentrationand desalinationof protein solutions by ultrafiltration, Chem. Eng. Sci., 57 (2002) 693. [6] E. Iritani, Y, Yukai and T. Murase, Separation of binary protein mixtures by ultrafiltration,Filtr. Sep., 34 (1997) 967. [7] S. Nakao, H. Osada, H. Kurata, T. Tsuru and S. Kimura, Separation of proteins by charged ultrafiltration membranes, Desalination, 70 (1988) 191. [8] A.G.Fane, C.J.D. Fell and A. Suki, The effect ofpH and ionic environment on the ultrafiltration of protein solutions with retentive membranes, J. Membr. Sci., 16 (1983) 195. [9] N.S. Pujar and A.L. Zydney, Electrostatic and electrokinetic interactions during protein transport through narrow pore membrane, Ind. Eng. Chem. Res., 33 (1994) 2473.
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[10] R.H.C.M. van Eijndhoven, S. Saksena and A.L. Zydney, Protein fractionation using electrostatic interactions in membrane filtration, Biotechnol. Bioeng., 48 (1995) 406. [11] S. Saksena and A.L. Zydney, Effect of solution pH and ionic strength on separation of albumin from immunoglobulin (IgG) by selective filtration, Biotechnol. Bioeng., 43 (1994) 960. [12] A.G. Fane, C.J.D. Fell and A.G. Waters, Ultrafiltration of protein solutions through partially permeable membranes: The effect of adsorption and solution environment, J. Membr. Sci., 16 (1983) 21 l. [13] M. Nystrom, P. Aimar, S. Luque, M. Kulovaara and S. Metsamuuronen, A fractionation of model proteins using their physiochemical properties, Coll. Sur£ A: Physiochem. Eng., 138 (1998) 185. [14] N.S. Pujar and A.L. Zydney, Electrostatic effect on protein partitioning in size exclusion chromatography and membrane filtration, J. Chromatography A, 796 (1998) 229. [15] A. Persson, A.S. Jonsson and G. Zacchi, Transmission of BSA during cross-flow microfiltration: influence of pH and salt concentration, J. Membr. Sci., 223 (2003) 11.
[16] S. Ikeda and K. Nishinari, Intermolecular forces in bovine serum albumin solutions exhibiting solid like mechanical behaviors, Biomacromolecules, 1 (2000) 757. [17] M. Elibol and D. Ozer, Response surface analysis of lipase production by freely suspended Rhizopus arrhizus, Process Biochem., 38 (2002) 367. [ 18] C.-J. Shieh, H.-F. Liao and C.-C. Lee, Optimization of lipase-catalyzed biodiesel by response surface methodology, Bioresource Technol., 88 (2003) 103. [19] D.R. Hamsaveni, S.G. Prapulla and S. Divakar, Response surface methodology approach for the synthesis ofisobutyl isobutyrate, Process Biochem., 36 (2001) 1103. [20] R.H. Myers and D.C. Montgomery, Response Surface Methodology, 2nd ed., Wiley, New York, 2002. [21] R. Chan, V. Chen and M.P. Bucknall, Ultrafiltration of protein mixtures: measurement of apparent critical flux, rejection performance and identification of protein deposition, Desalination, 146 (2002) 83. [22] C. Tanford, J.G. Buzzell, D.G. Rands and S.A. Swanson, The reversible expansion of bovine serum albumin in acid solutions, J. Amer. Chem. Sot., 77 (1955) 6421.
ELSEVIER
www.elsevier.com/locate/desal
Membrane application in proteomic studies: Preliminary studies on the effect of pH, ionic strength and pressure on protein fractionation M.M.D. Zulkali*, A.L. Ahmad, C.J.C. Derek School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal, Seberang Perai Selatan, Pulau Pinang, Malaysia Tel. +60 (4) 593-7788, ext. 6414; Fax." +60 (4) 594-1013; email: [email protected]
Received 29 September 2004; accepted 22 November 2004
Abstract
The effects ofpH, ionic strength and pressure on membrane-modulated protein fractionation were studied. Model protein, bovine serum albumin (BSA), was subjected to various pH, ionic strengths and pressures. The protein content and salt rejection in the retentate and flux were monitored. The design of the experiment was carried out with the aid of response surface methodology (RSM). A set of 20 designed experiments was carried out, and the data collected were analyzed using central composite experimental design in response surface methodology (RSM) by Design Expert, Version 5.0.7 (StatEase, USA). The response surface plot for fluxes was higher at lower ionic strengths and at high pH due to the shielding of charge and the electrostatic repulsion of protein with a similar charge while higher pressure reduced the flux through concentration polarization and expansion of adsorbed BSA on the membrane surface. Chloride rejection was highest at pH 8 and was independent of the salt concentration where the negative charge of BSA was stronger. It was observed that higher pressure seemed to favor rejection. Keywords: Ionic strength; Flux; pH; Response surface methodology
1. Introduction
Purification o f protein is often carried out using the chromatographic method, which is very specific, and a very pure fraction o f protein can *Corresponding author.
be obtained. However, the method is difficult to scale up, yielding a low throughput with an extremely high cost. Therefore, membranes are seen as an alternative that is cost effective and can be fine-tuned to achieve high productivity and purity at the same time [1-4]. Ultrafiltration
Presented at the conference on Membranes in Drinking and Industrial Water Production, L 'Aquila, Italy, 15-17 November 2004. Organized by the European Desalination Society.
0011-9164/05/$- See front matter © 2005 Elsevier B.V. All rights reserved doi: 10.1016/j.desal.2004.11.084
382
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
(UF) membranes can be used to separate protein using the same principle as chromatography as well as its natural separation, which is size dependent. If only the sizes of the pores are used as the means of separation with membranes, mostly 10-fold differences in molar masses of the proteins are needed because of the dispersed membrane pore sizes and fouling of the membranes. It is generally accepted that solutes whose molecular dimensions are small enough to permeate the membrane are substantially retained by the same membrane when larger solutes are present [5,6]. However, studies have shown that membrane filtration of proteins can also be drastically influenced by the nature of a solutesolute interaction [5-8]. These physicochemical interactions can and do occur between the membrane and solutes in the form of electrostatic charge, hydrophobic or even charge transfer. As a result, transmission of solutes through the membrane can either be manipulated by the physicochemical parameters such as pH and ionic strength or the operational parameters such as transmembrane pressure, system configuration, etc. The solute-solute interaction will determine the filtration rate and the rejection of solute when one or more proteins are ultrafiltered. Pujar and Zydney [9] showed that the rejection coefficient of bovine serum albumin (BSA) could be increased from about 0.2 to more than 0.995, simply by lowering the ionic strength. The dramatic increase in protein rejection is due to the strong electrostatic repulsion of the negatively charged protein from the pores of membrane. It is usually observed that proteins have the highest transmission at their isoelectric point (IEP) and lower transmissions when not at their IEP values [5,6,10,11]. For charged molecules such as proteinS, transport of rejected material back from the membrane surface to the bulk solution is not only dependent on the concentration gradient at the membrane interface but is
also driven by the electrostatic repulsion between the charged molecules. Owing to their charge repulsion, the protein will not be easily transmitted through a pore close to the size of the protein with a similar charge. It is mostly transmitted at its IEP where it has no charge. The effect of salt on filtration of protein can be seen as shielding of charges. Charged proteins, which normally do not penetrate the membrane, gain transmission through the membrane due to the shielding of charges. This shielding effect reduces the hydrodynamic diameter of a protein differing from its IEP. The shielding effect also naturally decreases the charge of the membrane. However, when proteins are at their IEP, normally the opposite effect is observed. The presence of salt decreases the retention of charged proteins and mostly increases the retention of neutral proteins. These effects have been shown by Fane and coworkers [8,12] and Nystrom et al. [13]. The hydrodynamic radius of the protein can thus be manipulated using salt. Pujar and Zydney [14] showed the influence of solution ionic strength when it was reduced from 150 to 1.5 mM and subsequently decreased the protein transmission by more than two orders of magnitude. The best fractionation was achieved when one of the proteins was at its IEP and the other one was retained due to the charge repulsion with the membrane as an example myoglobin and BSA at pH 4.8 [13]. The effect of the pH and salt concentration can be very profound. It is not limited to UF membranes and is found also in microfiltation (MF) membranes. As an example, BSA transmission can be as low as 40% when a MF membrane with 0.16 #m pores, which is definitely much larger than the molecule itself, is used [15]. In order to exploit effectively the electrostatic interaction for protein separation, it is important to evaluate the magnitude of these effects accurately. Noordman et al. [5] have shown that rejection and fluxes of BSA retention can be well
383
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
described by a model based on the MaxwellStefan theory of mass transfer with the nonidealities through the Carnahan-Starling equation of state for rigid spheres. The study was carried out by obtaining data for a single protein over a wide range of different environmental conditions, similar to those reported in literature elsewhere with respected to electrostatic interactions [7,9]. In the present study, an attempt was made to access the magnitude of pH, ionic strength and pressure on the protein and salt rejection by UF with the aid of response surface methodology (RSM).
valve
7 ~ 2 ~ Amicon
0- © Sd u b on reservoir
i Fig. 1. Ultrafiltration set-up. 0.45 0.4 0.35 ~"
2. Material and methods
2.1. Materials Protein solutions of appropriate concentrations were prepared. The model protein was BSA (Fraction V, Sigma Aldrich). BSA has a tertiary structure with an IEP of 4.9 and a molecular weight of 67,000 Da [16].The pH was adjusted with NaOH (Fluka, BioChemika) or HC1 (Fluka, BioChemika) and the ionic strength was adjusted via addition of solid crystals of NaC1 (Fluka, BioChemika). The protein solution was prepared no longer than 1 h before use. All solutions were prepared using deionized water. 2.2. Equipment and measurement The experiment was carried out in a feedbatch configuration as shown in Fig. 1. A circular flat-sheet membrane was placed on the bottom of an Amicon 8200 stirred cell supported by a porous plate. The effective area of the membrane was 28.27 cm 2. The cell contained a single blade stirrer driven by magnetic coupling at 500 rpm. The membrane cell was pressurized with nitrogen gas to the solution reservoir at the required pressure and that at the permeate side was at atmospheric. All experiments were carried out
0.3
~E g.. 0.25 g 0.2 o.15 IJ-
0.1 0.05 "-r--
1
2
3
4
Pressure (bar)
Fig. 2. Hydraulic permeability of polyethersulfone UF membrane (30 kDa). under ambient temperature. Mixing of the feed solution was accomplished by the stirring of the solution. An UF polyethersulfone flat-sheet membrane (PBTK), acquired from Millipore, with a 30,000 kDa molecular weight cut-off was used. The membranes were rinsed in deionized water for 1 h before use, and pretreatment of the membrane was carried out at 450 kPa for 1 h. The hydraulic permeability of the membrane was then determined (Fig. 2). Solution of 1 g/L of BSA was filled into the Amicon 8200 stirred cell. The loss of volume in the cell due to filtration was balanced with the similar concentration of BSA from the reservoir. The membrane was cleaned after each run. First, the membrane was rinsed with deionized water and then treated with 0.1 M NaOH solution for
384
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
30 rain at the same pressure as that used in the subsequent experiment. Then the membrane was flushed thoroughly with deionized water. The pure water flux was measured after the cleaning to evaluate the performance of the cleaning step. The flux was maintained within 5% difference from the original pure water flux. The permeate fluxes were measured gravimetrically. Concentrations of BSA both in permeate and concentrate were determined by UV absorption at 280 nm. The concentration of sodium chloride was determined through mercuric titration (chloride test kit, Hanna Instrument). The rejection of BSA turned out to be always larger that 99%, and therefore it was assumed to have 100% rejection in all the experiments.
2.3. Experimental design RSM is a useful technique in designing experiments for studying the effect of several factors influencing responses by varying them simultaneously and carrying out a limited number of experiments with the objective to optimize the response as oppose to the conventional method of optimization that involves varying one parameter at a time while keeping the others constant. RSM has been applied to optimize numerous biotechnological processes [17-19] due to its ability to include the interactions of several factors (or variables) that affect the response of a particular process. A central composite rotatable design (CCRD) was used to obtain a better understanding of the relationship between the variables of pH and ionic strength toward the response of flux, protein and ion retention. A 23 full factorial CCRD with five levels was used in the study which required 20 set of experiments. The variables and levels selected for the study on protein rejection were pH (3.5-8), NaC1 concentration (1.3-5 g/L) and pressure (2-4 bar). The range and the levels of the variables investigated in the research are given in Table 1. Table 2 shows the
actual experiment carried out for developing the model. The experimental data were analyzed by the response surface regression procedure to fit a second-order polynomial equation: k
k
Y = DO+ Z ~iXi + Z ~iiX? + Z Z i:1 i+1 i
~ijXiXj (1)
where y is the response, 13i the constant coefficient, and x~ the independent variable. The regression analysis, statistical significance and graphical analysis were done using Design Expert (Version 6.0, StatEase, USA) software. The statistical significance of the regression coefficients was determined by the Student t-test, the second-order model equation was determined by the Fischer's test and the proportion of variance explained by the model obtained was given by the multiple coefficient of determination, R 2.
3. Results and discussion
The effects of ionic strength, pH and pressure on flux and chloride rejection were studied using central composite design in RSM. The data analysis was preformed on a total of 20 sets of experiments consisting of eight factorial points, six axial points and six center points. The range chosen for the process variable were 3.5-8 for pH, 1.3-5 g/1 of NaC1 for ionic strength and 24 bar for pressure. A regression analysis was preformed to fit both the response function of flux and chloride rejection. Two equations were obtained through the program for each of the response. The model expressed by Eqs. (2) and (3), where the variables take their coded values, represents the percentage of chloride rejection (Ii,.) and flux (Yi) as a function of pH (x 0, ionic strength (x2) and pressure (x3). The coefficient of the second-order polynomial equation is given in Eqs. (2) and (3).
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
385
Table 1 Experimental domain Variables
Coded/range and level
x~ - - pH x2 - - NaC1 (g/L) x3- - Pressure (bar)
-2
-1
0
+1
+2
1.96 0.04 1.32
3.50 1.30 2.00
5.75 3.15 3.00
8.00 5.00 4.00
9.53 6.26 4.68
Table 2 Fxperimental data for RSM-CCRD analysis Run
xI
x2
x3
Flux (m3/mZ,h)
Chloride rejection (%)
1 2 3 4
0 0 0 0 - 1 0 0 -1 +1 -2 -1 0 +1 +1 0 +1 +2 0 0 -1
0 0 0 +2 +1 0 0 -1 -1 0 -1 0 +1 +1 0 -1 0 0 -2 +1
0 0 0 0 -1 0 0 +1 -1 0 -1 0 -t +1 -2 +1 0 +2 0 +1
0.129 0.136 0.101 0.133 0.145 0.132 0.197 0.081 0.178 0.131 0.168 0.147 0.135 0.139 0.140 0.144 0.127 0.156 0.140 0.080
3,19 1.56 9.09 t5.79 3.50 -3.79 7.69 -3.05 7.37 5.66 5.69 5.26 12.38 30.86 -4.20 3.18 13.55 3.82 -2.50 12.06
5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Yr = 3.72 + 3.58x 1 + 5.59x1 + 2.02x 3 + 2.79x 2 + 1 . 7 4 x ~ - 0.67x 2 + 2 . 4 7 x l x 2 + 1.81XlX 3 + 5.00xzx 3(2)
Y f : 0.14 + 5.679 × 10°3xl - 6.774 × 10-3x2 - 0.036x 3 + 0.015xax 2 + 0.015x 3
The fitness o f the model was expressed b y the coefficient o f determination, R 2. The R 2 for Eqs. (2) and (3) were 0.8665 and 0.5063, respectively. The R 2 value o f 0.8665 (86.65%) indicated the variability in the response o f the model where
(3)
13.35% o f the total variation was not explained b y the model [Eq. (2)], which m a y seem to be fairly good. However, Eq. (3) with an R 2 o f 0.5063 could not be explained variation by the model. The low coefficient o f determination
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M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
Table 3 ANOVA for model regression Source Flux model: Model residual Lack of fit Pure error Chloride rejection model: Model residual Lack of fit Pure error
Sum of squares
Degree of freedom
Mean square
F value
Probability level
1083.02 168.35 60.10 108.25
9 10 5 5
121.45 16.84 12.02 21.65
7.21
0.0024
0.56
0.7330
6.586 E-3 6.422 E-3 1.408 E-3 5.014 E-3
5 14 9 5
1.317 E-3 4.587 E-4 1.465 E-4 1.003 E-3
2.87
0.0547
0.16
0.9917
could be due to the constant changes of protein concentration along with the physicochemical properties as it is being concentrated, which influences the flux. Adding protein concentration as another variable may help to give a better model for prediction [22]. The ANOVA for both flux and salt rejection model is shown in Table 3. The rejection of salt observed during the protein purification is shown in the threedimensional presentation (correlation between pH, ionic strength and pressure on the chloride rejection) in Fig. 3a-c. It was observed that chloride rejection was very much dependent on the interaction ofpH, pressure and ionic strength. The chloride rejection was highest ranging from 3.73% to 28.05% at pH 8 (i.e., above its IEP), independent of salt concentration. It seems that the charge of the protein was the same as that of the chloride ions. Due to the concentration polarization and adsorption of the protein on the membrane [21], there was an accumulation of negative charges at the membrane that results in repulsion of chloride ions and attraction of the positive sodium ions. The net negative electrical potential on the membrane surface repelled the negatively charged chloride ions and thus increased its rejection. The rejection increased (with the highest increment of 18.01% rejection at pH 8, ionic strength at 5.00 g/L from a pressure
change of 2 bar to 4 bar), along with the operating pressure, partly due to the higher formation of concentration polarization at the membrane interface. On the other hand, at low pH (i.e., below the IEP) the chloride rejection was lower compared to rejection of the ion above the BSA's IEP due to the positively acquired charge of the adsorbed protein. The chloride ion, which was the counterion, then was attracted by the protein at the membrane interface. The chloride ion rejection was lowered. However, at lower operating pressures, the chloride rejection increased with lower ionic strength due to the reduction in charge shielding of the positive ion. The higher the ionic strength, the better the rejection of chloride, which was independent ofpH and pressure. The presence of a higher concentration of salts provided a better shielding of charge and thus reduced the retention effect on the membrane and concentration polarization. The effects ofpH, ionic strength and pressure towards flux are shown in Fig. 4a-c. It was seen that the fluxes behaved as expected from the electrostatic consideration point of view. The flux increased with decreasing ionic strength due to lower shielding of protein charges at low salt concentration. Therefore, the protein molecules strongly repelled each other, especially at the
387
M.M.D. Zulkali et al, / Desalination 179 (2005) 381-390
(a)
(b) 19.89 28,05
14.86
19.90
9.83
11.75
8 "~
3.6~
,~
4,7S
"~
-0.2,4
-4.5
_o
5.0 O0
B:lonic
ton
1.30 3.50 1,30 3.50
Fig. 3. Response surface plots of the effect of ionic strength and pH on chloride rejection at constant pressure. (a) 4 bar, (b) 3 bar, (c) 2 bar.
(c) 10.40 7.~ 5.57 3.1~ 0.7, "m o
B:ion
membrane surface where the concentration was high. The electrostatic repulsion increased the mass transfer of protein at the membrane interface back to the bulk solution. The concentration and thus, the osmotic pressure at the membrane surface were reduced and higher fluxes were observed. The effects of solution pH on fluxes can also be in Fig. 4a-c. The fluxes were higher at higher pH (i.e., above the protein's IEP). This
phenomenon was similar to that observed in the study on the effect of ionic strength. The higher the pH, the more negatively charged the protein and their electrostatic repulsion on the membrane surface was much stronger. The concentration polarization was reduced at the membrane surface and a higher flux was observed. However, at lower pH (below the protein IEP), the fluxes decreased with increased pressure due to the formation of concentration polarization on the surface of the membrane. It was reported that below the protein's IEP, the BSA molecules expanded with decreasing pH [22]. Owing to this effect, the mobility of the molecule also decreased, which led to more profound concentration polarization on the membrane surface. Therefore, the concentration polarization was enhanced and flux deteriorated. The increased of pressure seemed to affect the flux more when the solution pH was lower due to higher extend of concentration polarization at the membrane surface compared to when the pH was higher. The curvature of the response plots in Fig. 4a-e suggest a rapid decline in the flux in
388
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
(a)
(b) 0.174
0.16~
0.156
0.14
0.13~
0,1.~
0.12~
0.1
0.1C n-
C: 2,00
2.OO 3,50
3.50
Fig. 4. Response surface plots of the effect of pH and pressure on flux at constant ionic strength. (a) 1.3 g/L,
(c)
(b) 3.15 g/L, (c) 5.00 g/L. 0.I60 0,143 0
~E
C: Pressure (bar) O
0,1~7 -
125
0.10' 008 0.167 -
@
% 0,137x LL
0t06
C:
-
= D£sir~r] Pl:li!~i;
2 O0 350
m C- 2.000 A C + 4.009
0.076 -
response to the combined effect of pH between 3.5 to 5.75 and pressure between 2.5 bar to 3.5 bar. The decline in flux could be due to the conformational change of BSA influenced by the combined effect of pressure and pH. It was postulated that the molecule expanded and affects the transmission across the membrane surface. The effect of interaction of pH and pressure towards the flux at constant ionic strength of
I 3,50
I 4,63
I 5.75
I
i
6,88
8.00
A: pH
Fig. 5. Graph plotting interaction of pH and pressure towards flux at ionic strength of 3.15 g/L.
3.15 g/L is shown in Fig. 5. The flux decreased around 45% when pH was changed from 8 to 3.5 at high pressure, but at a low pressure the
M.M.D. Zulkali et al. / Desalination 179 (2005) 381-390
pressure lowered the flux by increasing the density of concentration polarization and at low pH (pH 3.5); the adsorption of protein on the membrane surface expanded and further aggravated the flux. The chloride rejection was highest at pH 8 and was independent of the salt concentration where the BSA protein had a similar charge as the chloride ion. It was also observed that higher pressure seemed to assist in the rejection.
B: I o n i c strength (g/L) 30.88
--
Desi~2.nPoints
i
B- 1.300 • 8÷ 5.000 2210
'~'
--
13.33 --
g
~ " ......
-[-
~...........
144v----....... T
0 4.57
•
~
~
389
-
• Q
-4.20 I .... :~,50
I
I
4.183
5,7~
I.... 6.88
I
References
8.00
A: pH
Fig, 6. Graph plotting interaction ofpH and ionic strength towards chloride rejection at a pressure of 3 bar. contrary was observed. It would seem that operating at high pressure and low pH was not feasible. The change of pH from an acidic to an alkaline condition seemed to have a more significant effect towards chloride rejection at high ionic strength (Fig. 6). The chloride rejection increased by more than 11% at pH 8 as compared to around 2% at low ionic strength. The results showed that the pH influence towards chloride rejection increased with the increase of ionic strength. Similar observations were made with regard to the interaction effects for other variables (Figs. 3a-c and 4a-c).
4. Conclusions It was clear that pH, ionic strength and pressure played important roles in affecting the physicochemical properties o f the protein and the transmission across UF membranes. The fluxes were higher at lower ionic strength and at higher pH (pH 8) owing to lower shielding o f charge and electrostatic repulsion o f protein molecules with similar charges. It was observed that higher
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