Protein Transport, Aggregation, and Deposition in Membrane Pores

Chapter 16

Protein Transport, Aggregation, and Deposition in Membrane Pores R. Chan and V. Chen

Downloaded by COLUMBIA UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0736.ch016

UNESCO Centre for Membrane Science and Technology, School of Chemical Engineering and Industrial Chemistry, University of New South Wales, Sydney, New South Wales 2052, Australia

Protein deposition on membrane surfaces and in pores can be probed using constant flux filtration experiments and electron microscopy. Protein precipitation near membrane pores are the result of pre -existing aggregates, shear enhanced multilayer protein adsorption, and aggregate formation and growth. To determine what hydrodynamic conditions and solution conditions are required to create protein aggregates in situ, fluxes were incrementally raised until a critical threshold was reached and membrane permeability suddenly decreased. Addition o f electrolytes was also used to change the protein interactions. Filtration below the critical threshold allows only low amount o f protein adsorption to be maintained. Above this threshold, multilayer protein growth is rapid and sufficient to reduce the pore size ten fold. Protein aggregates do not appear to occur until after the rapid rise in transmembrane pressure is observed, and the effect o f added electrolyte did not appear until a time period for protein rearrangement during filtration.

Membrane ultrafiltration and microfiltration form important separation processes for the separation and purfication o f proteins and other small colloids. Although membrane filtration has been addressed in the literature for more than 25 years, only recently have the dynamics o f protein transport, aggregation, and deposition near the membrane surface and pores have been accessible using new experimental techniques. During filtration through a partially retentive membrane, high concentrations o f protein may be confined on the membrane surface and a fraction of the solutes which reaches the surface w i l l undergo both diffusive and convective transport through tortuous pore structures. If adsorption reaches a significant level to prevent solute passage through the membrane yet pass solvent easily, a rapid build up o f solute at the membrane surface w i l l occur and fouling w i l l take place. The

© 1999 American Chemical Society

In Supramolecular Structure in Confined Geometries; Manne, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

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conditions leading to the onset o f protein adsorption due to concentration polarization, aggregation, and deposition on the surface and within the membrane is not well understood. Furthermore, many previous studies have ignored partially permeable membranes to simplify analysis o f the solute transport mechanisms to and from the membrane surface. However, controlling protein transport through ultrafiltration or microfiltration membranes allows optimal protein fractionation to be undertaken. In addition, controlling protein aggregation and deposition allows the maximum flux to be achieved without pore plugging and fouling. While there has been extensive studies on diffusive transport o f protein through membrane pores as well as passive protein adsorption, protein transport through U F and M F membranes is less well understood. Confinement o f a high solute concentration to the membrane wall, high local shear, and hindered passage through the membrane structure imposes limitations on solute passage even when the solute is much smaller than the pore size. Thus three potential mechanisms controlling protein transport and deposition in membrane pores should be considered: (a) protein adsorption, (b) protein aggregation (pre-existing and formed in situ in membrane pores), and (c) concentration polarization. The dynamics and control of these mechanisms are discussed. The consequence o f the local shear field and small pores is possible denaturation of the protein as they pass through the membrane. This denaturation increases the aggregation of the native protein, possibly to sizes large enough to plug the pores. There have been many studies on possible shear denaturation in processing of biological material, especially enzymes, with regards to pumping and exposure to the air-water interface. Charm and Wong showed that when the product o f the shear force and time exceeds 50 Pa s, measurable effect on the activity o f enzymes are observed (7). O n the other hand, Dunnill and co-workers reported no observable loss in enzymatic activity in their investigations (2). In membrane filtration units, proteins can be exposed to high shear forces (and concentrations) near pore walls for short periods as well as at low shear for long residence times due to pumping and recirculation. Convective deposition of protein aggregates generated by shear forces due to pumping was more extensively studied by Chandavarkar in the context of crossflow microfiltration (3). Using quasi-elastic light scattering and electrophoretic analysis, he showed that protein aggregates consisting of less than 1 % o f the total protein were detectable on the membrane surface and contributed significantly to pore occlusion. While protein aggregation due to bulk shear may be significant, the forces through small membrane pores are much higher but are o f short duration. Several studies have considered the potential for these shear forces to contribute to protein aggregation and fouling in membrane pores. Truskey et al. first investigated the possibility that membrane filtration may affect the protein conformation using low pressures and no stirring in a dead end cell (4). They did not find any significant protein conformation changes using circular dichroism ( C D ) . Subsequently, Franken et al. investigated fouling o f track etched



233 membranes with bovine serum albumin ( B S A ) (5). B S A is a prolate ellipsoid with approximate dimensions of 10 x 2.7 x 2.7 nm. B S A samples were examined by C D and Fourier Transformed Infrared (FTIR) after they have undergone shear, heating, and passage through a microfiltration membrane, and the secondary structures were compared. Using B S A prepared by the manufacturer using heat shock fractionation, rejection was observed with 0.2 um track etched polycarbonate membranes despite the high pore to protein size ratio. Rejection also increased with stirring rate and higher transmembrane pressures. These results indicated that proteins were able to effectively plug the pore structure, and that aggregation near the pore entrance was a possible mechanism. Franken et al. also observed that rejection did not occur when B S A prepared by Cohn cold ethanol fractionation was used in identical experiments. The F T I R and C D spectra of the B S A before and after filtration only showed small differences compared to the moderate changes shown by prolonged heating (2 hours at 70°C) and large changes due to high temperatures (5 min. at 100°C). This was not unexpected since B S A is considered a relatively rigid or "hard" globular protein, and the population o f shear-denatured protein may be small compared to the bulk solution. Thus evidence pointed to some population o f protein aggregates formed by both shear forces experienced during stirring, by shear experienced through the membrane pores, and by prior history. The effect is particularly magnified by low porosity (9.4%) track etched membranes. The average shear forces through the 0.2 um track etched membrane at 0.6 bar is calculated to be approximately 460 Pa but the residence time in the pores is less than 0.001 s. In the terminology of Charm and Wong, the shear-time product would be 0.22 Pa s, considerably less than the 50 Pa s they predicted to denature enzymes. This does not account for the possibility of high local shear gradient near the walls to cause protein denaturation. Hodgson compared multiple filtration o f B S A solution through highly porous alumina "Anopore" membranes (with 0.02 and 0.2 um poresize) (6). Hodgson investigated both the possibility o f pre-existing protein aggregates and those formed by confinement under shear in membrane pores (6). These membranes have the advantage o f the high porosity and uniform capillary pores. Particle sizing of the raw protein solution and protein recovered from the surface o f the 0.02 um membranes were compared in the 200 - 2000 nm range. The aggregates on the surface o f the membranes contained a larger proportion o f large aggregates than in the raw solution. He found that the reduction of flux and the changes in protein transmission followed neither a simple internal deposition nor an external cake deposition mechanism. Instead he proposed that the following stages of fouling occurred. First, a rapid monolayer adsorption in the pores occurs, with some pre existing aggregate being deposited from bulk solution. Second, a more substantial aggregate layer formed due to aggregate growth through protein deposition on their surface. Eventually, the growth o f the aggregates forms an even cake or "sheet-like" layer which parallels the onset of rejection (6).



234 Bowen and Gan observed, that unlike normal passive protein adsorption, filtration o f dilute B S A proteins in Anopore membranes indicated a continuous protein deposition with a maximum layer thickness o f 55 nm on the walls o f the 0.2 um diameter walls (7). This is in contrast with monolayer passive adsorption o f 5 10 nm observed in the absence o f filtration. Thus shear and confinement in membrane pores thus result in both multilayer protein adsorption as well as aggregate initiation or growth. K e l l y et al. confirmed that the different types of commercially available B S A showed that the rate o f flux decline was most rapid for proteins prepared by the heat shock fractionation and those designated fatty acid free (8). Prefiltration o f their protein solutions slowed the flux decline but did not prevent continuous flux decline. K e l l y et al added iodoacetic acid which caps the free sulfhydryl group on each B S A molecule in an attempt to limit further aggregation of the protein solution during filtration (9). Their results showed that capping o f sulfhydryl group followed by prefiltration to remove the existing aggregates resulted in a solution which exhibited very low flux decline. These results support the preliminary evidence by Franken et al. and Hodgson that both pre-existing aggregates and aggregate formation play a role in the dramatic flux decline observed in protein filtration (5,6). Attempts to directly observe the protein deposits on membrane surfaces have been conducted using field emission electron microscopy. Small volumes of protein solutions were filtered at various constant pressures, and the initial protein deposits were observed. K i m et al. showed that both pressure and membrane morphology resulted in more evident protein aggregate formation on the surface o f the membrane (10). Formation o f smooth cake like layers were observed at lower pressure while an increase o f from 100 to 200 kPa induced protein aggregates to appear on top o f 100 k D molecular weight cut off polysulfone membranes (77). Changes in p H and stirring did not appear to significantly affect the appearance o f the initial protein deposit. While the appearance o f protein aggregates is important in explaining the apparent rejection o f solutes many times smaller than the pore size, the onset of both the irreversible flux decline and rejection was identified as a function o f achieving some critical condition near the membrane pore walls. More recently, additional techniques have been used to probe the critical conditions to initiate irreversible multilayer protein deposition. Anecdotal evidence had often suggested that moderate flux during initial startup o f membrane filtration and operation can reduce the severity o f fouling. Turker et al. and others controlled the permeate flow rather than use a fixed transmembrane pressure to control deposition to the surface (12-14). Thus much more consistent hydrodynamic conditions can be applied during the filtration process whereas constant pressure filtration resulted in a continuing decreasing convective force on the different layers of deposition. Since the deposition of the particles depend on the balance between convective drag and axial convection, the concept o f "critical flux" has been used to describe the



235 hydrodynamic conditions to achieve little or no deposition o f colloidal particles on the membrane surface. A demonstration of this concept was shown by Field et al. with dodecane emulsion and yeast particles (13,14). Field et al. were able to maintain filtration without flux decline and showed that once a threshold flux was exceeded, deposition rapidly took place (13,14). Using a series o f increasing flux steps, Madaeni showed that there was a sharp transition between "nonfouling" or "subcritical" flux and a rapidly fouling regime, even a complex fluid such as activated sludge (75). For smaller particles, Chen et al showed that the transition from concentration polarization to irreversible cake deposition can be detected by the same means using 12 nm silica particles (76). A t a fixed, constant flux, the transmembrane pressure is constant when only concentration polarization is present as it is established rapidly; however, at the fouling threshold, a rapidly increasing transmembrane pressure occurs. Reversibility o f this deposition depends on the how much excess pressure is applied. Rather than a "critical flux", Aimar and co-workers have considered the conditions for irreversible deposition to occur when both the colloidal interactions between particles as well as the convective forces bringing the particles into contact reaches some threshold level (77). This threshold depends on particle size, concentration, and interaction parameters between the colloidal particles such as V a n der Waals forces and electrostatic repulsion. The regimes favorable for particle deposition were mapped out by Aimar and co-workers based on the hydrodynamic factors (crossflow and flux) and the potential barrier V which represent the colloidal interactions, using a factor W = 1 + (V /5) as a criterion for determining i f a particle w i l l be retained on a membrane surface (7 7). High W values indicate low or negligible depositions and the crossover between nonfouling and fouling regimes is delineated by where the Sh ~ Pe, ie mass is dominated by convection and not by colloidal interaction V . B

D

B

D

B

The Sherwood number is defined as a function o f both the Peclet number and the potential barrier V , B

Sh = ,e

p

(W -\)e-' +^ (\-e-

Protein Transport, Aggregation, and Deposition in Membrane Pores

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