Biotechnology Advances 24 (2006) 482 492 www.elsevier.

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Research review paper

Membrane processes in biotechnology: An overview

Catherine Charcosset
Laboratoire d'Automatique et de Gnie des Procds, UMR CNRS 5007, UCBLyon 1, ESCPE-Lyon, 43 Bd du 11 Novembre 1918, 69 622 Villeurbanne Cedex, France Received 10 October 2005; received in revised form 12 March 2006; accepted 20 March 2006 Available online 9 May 2006

Abstract Membrane processes are increasingly reported for various applications in both upstream and downstream technology, such as the established ultrafiltration and microfiltration, and emerging processes as membrane bioreactors, membrane chromatography, and membrane contactors for the preparation of emulsions and particles. Membrane systems exploit the inherent properties of high selectivity, high surface-area-per-unit-volume, and their potential for controlling the level of contact and/or mixing between two phases. This review presents these various membrane processes by focusing more precisely on membrane materials, module design, operating parameters and the large range of possible applications. 2006 Elsevier Inc. All rights reserved.
Keywords: Ultrafiltration; Microfiltration; Membrane bioreactor; Membrane chromatography; Membrane contactor

Contents 1. Introduction . . . . . . . . . . . 2. Ultrafiltration and microfiltration 3. Membrane bioreactors. . . . . . 4. Membrane chromatography . . . 5. Membrane contactors . . . . . . 6. Conclusion . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482 483 485 486 488 489 490

1. Introduction Membrane processes are increasingly being used in reaction, clarification, and recovery schemes for the production of molecules, emulsions and particles. Membrane systems take advantage of their selectivity, high surface-area-per-unit-volume, and their potential
Tel.: +33 4 72 43 18 67; fax: +33 4 72 43 16 99. E-mail address: charcosset@lagep.univ-lyon1.fr. 0734-9750/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2006.03.002

for controlling the level of contact and/or mixing between two phases. They are very well suited to the processing of biological molecules since they operate at relatively low temperatures and pressures and involve no phase changes or chemical additives, thereby minimizing the extent of denaturation, deactivation, and/or degradation of biological products (Zeman and Zydney, 1996). This review presents various membrane processes, the established ultrafiltration and microfiltration, and emerging processes such as membrane

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bioreactors, membrane chromatography, and membrane contactors for the preparation of emulsions and particles. Ultrafiltration and microfiltration are commonly used to recover macromolecules and retain suspended colloids and particles, and are being integrated into both upstream and downstream processes. A large range of ultrafiltration and microfiltration applications is reported to concentrate proteins, exchange buffer systems, clarify suspensions for cell harvesting, and sterilize liquids to remove viruses and bacteria. Other membrane processes include membrane bioreactors, where enzymes, microorganisms or antibodies are suspended in solution and compartmentalized by a membrane in a reaction vessel or immobilized within the membrane matrix itself. Membrane chromatography is used as an alternative to conventional resin-based chromatography columns, for a large range of chromatographic purification schemes, including ion-exchange, hydrophobic, reversed-phase, and affinity chromatography. Finally, membrane contactors involve using a pressure to force the dispersed phase to permeate through a membrane into the continuous phase, for the preparation of emulsions and various types of particles, as w/o emulsions, o/w emulsions, and polymeric particles. This review presents these membrane processes by focusing more precisely on membrane materials, module design, operating parameters and the large range of possible applications in the biotechnology field. 2. Ultrafiltration and microfiltration Ultrafiltration and microfiltration are well-known membrane separation processes (i.e. Zeman and Zydney, 1996; McGregor, 1986). Module configurations include hollow fiber, tubular, flat plate, spiral wound and rotating devices. The two standard modes of operation are deadend and cross-flow configurations (Fig. 1). In the crossflow mode, the fluid to be filtered flows parallel to the membrane surface and permeates through the membrane due to a pressure difference. The cross-flow reduces the formation of the filter cake to keep it at a low level. A very common application of ultrafiltration in downstream processing is for product concentration (i.e. buffer or solvent removal). Buffer exchange and desalting can be done using diafiltration to wash the initial buffer out and to replace it with a new buffer. The most common application of microfiltration is sterile filtration (bacterial removal) prior to final formulation of many products, and in the initial clarification of fermentation broths to remove the suspended cell mass and other particulate debris. Sterile filtration is performed in

Feed

(a)

Membrane

Permeate Feed Retentate

(b)
Membrane

Permeate
Fig. 1. Comparison between: (a) dead-ended and, (b) cross-flow configurations.

a dead-end configuration using 0.2 m pore size membranes that have been validated for the absolute removal of Brevundimonas diminuta (Kuriyel and Zydney, 2000). However, such sterilizing filters can pass very small microorganisms under some process conditions. Therefore, some users employ 0.1 m pore size membranes to provide enhanced sterility assurance in pharmaceutical processes (Sundaram et al., 1999). An important application of microfiltration is virus removal from cell cultures, to separate viruses that range in size from about 12 to 300 nm from proteins of 4 to 12 nm (Aranha-Creado et al., 1998; Liu et al., 2000; Van Reis and Zydney, 2001). Mammalian cell lines are frequently used in the production of recombinant proteins which have therapeutic, prophylactic or diagnostic applications. However, cell lines may be contaminated with virus or virus-like particles. In addition to endogenous contaminants, viruses may also be introduced by addition of supplements and other constituents introduced into the fermentation process, or through handling and other manipulations during processing. Membrane manufacturers are developing membrane filters with increasing resolution for virus-protein separation (Van Reis and Zydney, 2001). This is of significant importance to the biotechnology industry because incidents of parvovirus contamination of cell cultures may occur. Parvoviruses are particularly difficult to remove, as they are both small (about 20 nm diameter) and highly resistant to many thermal and chemical methods of inactivation. To ensure that virus removal is consistent with validation studies, membrane integrity is monitored both pre- and post-use. Membrane filtration is also particularly well suited in antibiotic production. Most antibiotics, such as

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benzylpenicillin (penicillin G), erythromycin and medmycin, are widely used and also serve as raw materials for semisynthetic antibiotics. They are produced by fermentation and recovered from their broths via the conventional steps: filtration (removal of biomass), solvent extraction (isolation and purification) and subsequent crystallisation (polish). Membrane filtration is used for the initial clarification of these fermentation broths (Nabais and Cardoso, 1999; Adikane et al., 1999; Moro et al., 2001; Brites Alves et al., 2002). One of the main advantages of membrane systems for the initial recovery of antibiotics from a fermentation broth is the ability to obtain very high yield using a combined filtration and diafiltration process. Complete retention of the cells and particulate matter can be achieved using membranes with pore sizes up to 0.20.45 m, and essentially complete passage of the antibiotics can be achieved as long as the nominal membrane molecular cut-off is greater than about 20,000 g/mol (Zeman and Zydney, 1996). Ultrafiltration may also be used to remove emulsifiers in antibiotic broths before solvent extraction to avoid emulsification and to improve extraction efficiency (Li et al., 2004). An example in this field is the work of Nabais and Cardoso (1999) on the purification of benzylpenicillin filtered broths obtained from fermented broths by ultrafiltration with diafiltration. This study was done using an ultrafiltration pilot installation with a tubular membrane, from Paterson Candy International Ltd. Membranes with molecular weight cut-offs of 8000 (polysulfone), 20,000 (polysulfone) and 100,000 (PVDF) were tested. Proteins, colored substances and other impurities were successfully removed, giving high benzylpenicillin recovery in the permeate, while demonstrating that ultrafiltration may be an alternative to the use of flocculants and anti-emulsion agents to obtain good phase separation in benzylpenicillin solvent extraction. Microfiltration and ultrafiltration have numerous applications in the biotechnology field. One may cite the following examples: recovery of heterogeneous immunoglobulins (IgG) from transgenic goat milk by microfiltration (Baruah and Belfort, 2004), concentration and purification of recombinant Brain-Derived Neurotrophic Factor (rBDNF) inclusion bodies from E. coli cell suspensions by cross-flow microfiltration and diafiltration (Schutyser et al., 2002), recovery of naturally glycosylated therapeutic proteins produced from animal cell cultures by microfiltration (Vogel and Kroner, 1999), recovery and purification of yeast alcohol dehydrogenease (ADH) from bakers' yeast as typical of downstream processing for the extraction of an intracellular enzyme product (Levesley and Hoare, 1999). The work of Baruah

and Belfort (2004) on the optimization of monoclonal antibody recovery from transgenic goat milk by microfiltration is an interesting example. The optimization involved varying pH, transmembrane pressure, milk feed concentration, membrane module type, and axial velocity. Operation in the pressure-dependent regime at low uniform transmembrane pressures using permeate circulation in co-flow, at the pI of the protein (9 in this case) is shown to increase IgG recovery from less than 1% to over 95%. Such methodology is generally applicable to the recovery of target proteins found in other complex suspensions of biological origin. Much effort is still being devoted to developing new membrane modules with improved mass-transfer characteristics for ultrafiltration and microfiltration processes. This includes rotating disk filters (Jaffrin et al., 2004; Lee et al., 1995), cylindrical Taylor vortex devices (Parnham and Davis, 1995), conically shaped rotors (Vogel and Kroner, 1999), and helical coiled Dean vortex systems (Luque et al., 1999; Schutyser et al., 2002). Dean vortex devices have very high mass-transfer rates, owing to the presence of centrifugal flow instabilities. These devices show significant increases in protein transmission and capacity, although fouling remains a problem in many applications. An alternative approach is to use highfrequency back-pulsing to continually clean the membrane surface (Levesley and Hoare, 1999). High-frequency back-pulsing was shown to improve flux, reduce fouling and increase protein transmission in the purification of conjugated vaccine products (Meacle et al., 1999). Other techniques to improve ultrafiltration and microfiltration performances include pulsative flow, gas sparging, electric fields, ultrasonic fields, and combined electric/ ultrasonic fields (Wakeman and Williams, 2002). In this field, one may cite the work of Jaffrin et al. (2004), which compares the effects of various hydrodynamic parameters on the permeate flux provided by two different dynamic filtration systems using the same membrane materials and the same fluids. Tested systems were two home-made rotating disk modules and a VSEP pilot with a circular vibrating membrane. The flux was found to be mainly governed by the maximum shear rate and not by details of internal flow and can be increased to very high levels by increasing rotation speed or vibration amplitude or by equipping the disk with large vanes. These results can be used for scaling up industrial systems. Another example in this field is the work of Schutyser et al. (2002) who used Dean vortex microfiltration with controlled centrifugal instabilities (Dean vortices produced in helical flow) to improve cross-flow microfiltration and diafiltration for the concentration and purification of rBDNF inclusion bodies from E. coli cell suspensions. For the

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microfiltration experiments with the feeds containing cell and homogenate suspensions, improvements in flux of about 50 and 70%, respectively, were obtained with the helical module as compared with that obtained with the linear module. For diafiltration with the homogenate suspensions as feed, solute transport was from 100% to 40% higher after 40 and 100 min, respectively, with the helical module as compared with that obtained with the linear module. Continued improvements in understanding the effects of solution environment on molecules, particles retention, and fouling (Aimar et al., 1993) have also led to further enhancements in ultrafiltration and microfiltration performances. For example, a number of recent studies have demonstrated that it is possible to control the rate of protein transport through membranes by adjusting the solution pH or the ionic strength (van Reis et al., 1997; Nystrom et al., 1998; Baruah et al., 2005). The process has to be operated at the pI of the transmitted protein and far away from the pI of the retained protein. To enhance the separation, the ionic strength is kept low so that the thickness of the diffuse double layer of the charged solute is pronounced, leading to high retention, whereas the uncharged solute readily permeates the membrane. These electronic interactions can also be exploited for protein separation using a process known as High Performance Tangential Flow Filtration (HPTFF). For example, van Reis et al. (1999) were able to achieve 99-fold purification of an antigen-binding fragment of a monoclonal antibody (Fab) from BSA by operating the membrane process near the isoelectric point of the BSA and using a positively-charged membrane to obtain very high rejection of the positively-charged Fab. 3. Membrane bioreactors Membrane bioreactors have been reviewed previously (i.e. Belfort, 1989; Belfort and Heath, 1993; Cheryan and Mehaia, 1986; Giorno and Drioli, 2000; Giorno et al., 2003). They are alternative approaches to classical methods of immobilizing biocatalysts such as enzymes, microorganisms and antibodies, which are suspended in

solution and compartmentalized by a membrane in a reaction vessel or immobilized within the membrane matrix itself. In the first method, the system might consist of a traditional stirred tank reactor combined with a membrane-separation unit; in the second method, the membrane acts as a support for the catalyst and as a separation unit (Fig. 2). The biocatalyst can be flushed along a membrane module, segregated within a membrane module, or immobilized in or on the membrane by entrapment, gelification, physical adsorption, ionic binding, covalent binding or cross-linking. The advantages of immobilizing enzymes are reported to include increased reactor stability and productivity, improved product purity and quality, and reduction in waste (Giorno and Drioli, 2000). Many studies are oriented to the investigation of operating conditions and optimization of the various properties of these membrane bioreactors. The efficiency of the overall system depends on the biochemical (e.g., catalytic activity, reaction kinetics, concentration, viscosity of substrate and product, immobilization stability), geometric parameters (e.g., membrane configuration, morphology and pore size distribution) and hydrodynamics parameters (such as transmembrane pressure and flow velocity) (Giorno et al., 2003). Immobilized biocatalyst membrane reactors are frequently used in a hollow-fiber configuration because of their high packing density (large surface area per unit volume of reactor space). Membrane bioreactors have been used for the production of aminoacids, antibiotics, anti-inflammatories, anticancer drugs, vitamins, optically pure enantiomers and isomers, etc. For example, membrane bioreactors have been reported for the synthesis of lovastatin with immobilized Candida rugosa lipase on a nylon support (Yang et al., 1997), the production of diltiazem chiral intermediate with a multiphase/extractive enzyme membrane reactor (Lopez and Matson, 1997), the synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase (Goulas et al., 2004), the production of a derivative of kyotorphin (analgesic) in

(a)
Reactants Reactor Permeate Membrane Permeate Membrane Immobilized Enzyme Reactants Retentate

Fig. 2. Membrane bioreactor configurations: (a) reactor combined with a membrane operation unit, (b) reactor with the membrane active as a catalytic and separation unit.

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solvent media using -chymotrypsin as catalyst and alumina mesoporous tubular support (Belleville et al., 2001), and biodegradation of high-strength phenol solutions by Pseudomonas putida using microporous hollow fibers (Chung et al., 2005). An example of this approach is the work of Nakajima et al. (1989) who developed a forced-flow membrane enzyme reactor, in which the enzyme is immobilized on porous ceramic membranes. By attaching the enzymes to the porous membrane surface, the mass transfer was improved by utilizing convection rather than diffusion, and the convection was not limited by the significant pressure drops found in enzyme-immobilized bead-filled column reactors. A 10fold higher productivity was observed in their system as compared to a conventional column reactor in which the enzyme was immobilized on beads. Immobilized whole cell membrane bioreactors have also been tested successfully (Belfort and Heath, 1993). Membrane bioreactors for immobilized whole cells provide an environment for increased cell densities so as to produce higher product titre. The cells are perfused via a membrane with a steady continuous flow of medium and supplied with oxygen and nutrients while wastes and desired products are removed. The cells are usually retained in the bioreactor through a membrane barrier. Many membrane configurations have been tested (such as flat sheet and rotating bioreactors), although the hollowfiber configuration is a particularly interesting one. Cells are either grown in the extracapillary space with medium flow through the fibers, or grown within the fibers with medium flow outside or across the fibers. It was shown that a mass transfer limitation for oxygen and glucose was obtained and that even, well-defined spacing between the fibers was a means of reducing this phenomenon (Heath and Belfort, 1988). Other geometries were proposed, such as hollow fibers inserted within another to grow the cells in the annulus between the two fibers (Custer, 1988). A proper choice of inner and outer fibers diameters can limit the distance needed for diffusion of medium components (oxygen, glucose, glutamine and other nutrients) to approximately 50 m for the furthest cells. A comparison of the conventional and concentric reactors for antibodies production from hybridoma cells showed that a much high concentration of cells can be maintained in the concentric reactor along with higher specific productivity and cell viability. For membrane bioreactors with immobilized enzymes, an important aspect is the chemistry of enzyme immobilization. It is often accomplished either directly on the membrane or via spacer arm, often through the -amino functionality of lysine residues on the protein (Butterfield et al., 2001). Although immobilization of

enzymes generally enhances their stability, one major disadvantage of random immobilization of enzymes onto polymeric microfiltration type membranes is that the activity of the immobilized enzyme is often significantly decreased because the active site may be blocked from substrate accessibility, multiple point-binding may occur, or the enzyme may be denatured. Different approaches are developed in order to accommodate site-specific immobilization of enzymes with different structural characteristics, as gene fusion to incorporate a peptidic affinity tag at the N- or C-terminus of the enzyme; post-translational modification to incorporate a single biotin moiety on enzymes; and site-directed mutagenesis to introduce unique cysteines to enzymes (Butterfield et al., 2001). Membrane bioreactors have been introduced over 30 years ago, and until now their main industrial applications have been for wastewater treatment (e.g., industrial, domestic and municipal) (i.e. Yang et al., 2006). In the biotechnology field, membrane bioreactors have obtained only limited success. The main technological difficulties in using membrane bioreactors on an industrial level are related with rate-limiting aspects and scale-up difficulties of this technology, together with the life-time of the enzyme, the availability of pure enzyme at an acceptable cost, the necessity for biocatalysts to operate at low substrate concentrations and microbial contamination. 4. Membrane chromatography Adsorptive membranes have been studied for nearly two decades as an alternative to conventional resin-based chromatography columns (Roper and Lightfoot, 1995; Charcosset, 1998; Klein, 2000; Ghosh, 2002). The benefit of adsorptive membranes is the shorter diffusion times than those obtained in resin-based chromatography, as the interactions between molecules and active sites on the membrane occur in convective through-pores, rather than in stagnant fluid inside the pores of an adsorbent particle (Fig. 3). For this reason, adsorptive membranes have the potential to maintain high efficiencies both at high flow-rates and for use of large biomolecules with small diffusivities. Their use is also accompanied by reduced protein degradation and denaturation. Chromatographic membranes have been used in a variety of configurations, as staked membranes, hollow fibers, spiral wound membranes and a variety of adsorptive mechanisms, such as ion-exchange, hydrophobic, reversed-phase, and affinity based procedures. Ion-exchange membranes have been investigated with strongly acidic groups (sulfonic acid), strongly basic (quaternary ammonium), weakly acid (carboxylic acid), and weakly

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Particles system

Membrane system

L 3 L L 3 S 2 Limitations: 1. Film diffusion 2. Pore diffusion 3. Binding kinetics L 1 S

L 1

Limitations: 1. Film diffusion 3. Binding kinetics

Fig. 3. Comparison between: (a) membrane chromatography and, (b) gel bead chromatography. L: ligand, S: solute.

basic (diethylamine) types. Affinity membranes have been tested with a large range of ligands such as immunoaffinity ligands, Protein A and G, low-molecular-mass ligands (Cibacron Blue, histidine, tryptophan) and other ligands (peptide, Cu2+) (Ghosh, 2002). Membrane materials tested for chromatographic applications include cellulose, polysulfone, polyamide, hydrazide, and composite membrane such as blend of polyethersulphone and polyethylene oxide coated on all surfaces with a covalently bound layer of hydroxyethylcellulose (Charcosset, 1998). Commercial membranes include flat sheet systems (Pall, New York), membrane stacks (Sartorius, Gttigen, Germany), radial flow cartridges (CUNO Europe, Cergy-Pontoise, France) and hollow-fiber modules (Kinetec System Inc., St Louis, MO). Ion-exchange membranes (cation and anion exchange) are available as well as affinity membranes (protein A and Cu2+ as ligands). Purification procedures using chromatographic membranes have been reported for a wide variety of compounds, such as proteins (monoclonal antibody, serum antibody, serum albumin, enzymes, etc.), DNA and viruses. Examples of applications are the use of thiophilic membranes for the purification of monoclonal antibodies from cell culture media (Finger et al., 1995), immobilized L-histidine in hollow-fiber membranes for the separation of immunoglobulin G from human serum (Bueno et al., 1995), affinity membranes for the separation of MBP

fusion proteins (Cattoli and Sarti, 2002), ion-exchange membranes for the isolation of antibacterial peptides from lactoferrin (Recio and Visser, 1999), cation-exchange membranes for the purification of alphaviruses (Karger et al., 1998), anion-exchange membranes for the adsorption of DNA (Charlton et al., 1999), and strong anionexchange membranes for reduction of endotoxin in a protein mixture (Belanich et al., 1996). The first paper to be published in the field of membrane chromatography by Brandt et al. (1988) is particularly interesting. A hollow-fiber device was proposed for purification of fibronectin from blood plasma and purification of IgG using hollow-fiber membrane-supported protein A. The high throughput rate and the efficient ligand use of this device permitted rapid bindelute cycle times. Because the volume of a typical agarose affinity system was 1001000 times that of the affinity-membrane device, the membrane device required only about 0.1% as much ligand to handle the same throughput at the same mass transfer efficiency. The work of Belanich et al. (1996) provided an example of a successful application in the pharmaceutical manufacturing. The scale-up of strong anion-exchange membrane adsorbers that remove endotoxin from bacterial extracts while preserving enzyme activity in the protein mixture was demonstrated. The endotoxin removal procedure was directly adapted from the small-scale Q-100 MA cartridge (Sartorius Corporation, 100 cm2 working surface area) to the large-scale Q-

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550 MA sheets (5500 cm2 working surface area). The characteristics of endotoxin removal, protein absorption, and photolyase purification were similar in the two systems. One of the major limitations with membrane chromatography is non-uniform flow distribution across the membrane, due to the large diameter-to-length ratio of the modules. This limitation was also pointed out by process mathematical modelling, which had to take into account the system dispersion curve to obtain an exact comparison between calculated and experimental concentrations (Sarfert and Etzel, 1997). This may represent a significant problem in many cases, reducing the membrane efficiencies to the level of packed beds. However, proper design of flow distributors was shown to reduce this problem (Ghosh, 2002). The membrane chromatography technique has certainly not obtained the expected success (i.e., membrane discs for chromatography are no longer provided by Millipore Inc.). The reason is probably due to the reticence of potential users for this new technology. In addition, membranes for chromatography are particularly attractive for preparative chromatography, as initially developed by Sepracor Inc. (Brandt et al., 1988), to purify large amounts of molecules. In this regard, hollow fibers (Brandt et al., 1988) are particularly well suited, more than flat sheet membrane modules. Finally, membranes for analytical chromatography present less advantage over classical chromatographic supports than those obtained for preparative chromatography. 5. Membrane contactors Membrane contactors have recently experienced increasing interest (Drioli et al., 2005). They have been reported, for example, for the preparation of emulsions (membrane emulsification) (Joscelyne and Trgrdh, 2000; Charcosset et al., 2004) and for the preparation of precipitates (Jia et al., 2003; Chen et al., 2004). The term

membrane contactor can be defined as the connection of two phases A and B through membrane pores. The membrane contactor involves using a pressure to force a dispersed phase A to permeate through a membrane into a continuous phase B, which flows tangentially to the membrane surface (Fig. 4). Membrane pores therefore behave as parallel capillaries for the introduction of phase A into phase B. There may either be no reaction between the two phases (membrane emulsification) or a reaction may occur between the two phases (preparation of precipitates). In case of membrane emulsification, droplets grow at the pore outlets until, upon reaching a certain size, they detach. This is determined by the balance between the drag force on the droplet from the flowing continuous phase, the buoyancy of the droplet, the interfacial tension forces and the driving pressure (Joscelyne and Trgrdh, 2000). The final droplet size and size distribution are determined by the pore size and size distribution of the membrane and also by the degree of coalescence, both at the membrane surface and in the bulk solution. A typical experimental set-up for a membrane emulsification process includes a tubular microfiltration membrane, a pump, a feed vessel, and a pressurized (N2) oil container. The oil phase (to be dispersed) is pumped under gas pressure through the membrane pores into the aqueous continuous phase which circulates tangentially to the membrane surface. Membrane emulsification has been developed during the past 15 years (Joscelyne and Trgrdh, 2000; Charcosset et al., 2004). The distinguishing feature is that the resulting droplet size is controlled primarily by the choice of the membrane and not by the generation of turbulent droplet break-up. The apparent shear stress is lower than the conventional emulsification systems, because small droplets are directly formed by permeation of the dispersed phase through the micropores, instead of disruption of large droplets in zones of high-energy density. Besides the possibility of using shear-sensitive

Continuous phase Tangential flow Membrane

Droplets

Dispersed phase Permeation under applied pressure

Fig. 4. Schematic diagram of the membrane emulsification process.

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ingredients, emulsions with narrow droplet size distributions can be produced. Furthermore, membrane emulsification processes allow production of emulsions at lower energy input (10 4 10 6 J/m 3 ) compared to conventional mechanical methods (10 6 10 8 J/m 3 ) (Altenbach-Rehm et al., 2002). Single (w/o or o/w) and multiple (w/o/w, o/w/o) emulsions, with various droplet sizes ranging from 0.8 to over 100 m, and a typical coefficient of variation of 10 15%, have been prepared. Various microspheres with diameters from 2 to 100 m have been synthesised, by combining the membrane emulsification process and subsequent suspension polymerization (Ma et al., 2003), subsequent solvent evaporation (Liu et al., 2005), or by using the droplet swelling method (Omi et al., 1997). Particles prepared primarily by membrane emulsification also include metal solder particles, embedded TiO2 microcapsules, bichromal particles, solid microcarriers (Vladisavljevi and Williams, 2005) and solid lipid nanoparticles by the cooling of the lipid emulsion formed by membrane emulsification to room temperature (Charcosset and Fessi, 2005b). Microporous membranes used by different investigators for membrane emulsification include for example SPG (Shirasu Porous Glass) membranes, coated -alumina or zirconia, anodic porous alumina, polypropylene, polyamide, and polytetrafluoroethylene (PTFE) membranes (Joscelyne and Trgrdh, 2000). Ma et al. prepared a large range of particles, such as uniform polyurethaneurea-vinyl polymer microspheres of about 20 m, using a Shirasu Porous Glass (SPG) membrane emulsification technique and subsequent radical suspension polymerization (Ma et al., 2003). For this, a mixture of a 40% urethane prepolymer solution of xylene and a vinyl monomer containing an initiator was permeated through the uniform pores of the SPG membrane into a continuous phase containing a stabilizer to form uniform droplets. The droplets were then allowed to stand for chain extension at room temperature with di- or triamines for 2 h in the absence or presence of ethyl acetate, followed by suspension polymerization at 70 C for 24 h. Other applications have been reported, which involve a reaction between phase A passing through the membrane pores with phase B flowing tangentially to the membrane surface, i.e. for the preparation of nanosized BaSO4 and CaCO3 particles (Jia et al., 2003; Chen et al., 2004), and polymeric nanoparticles, by interfacial polymerization or nanoprecipitation reaction between an organic and an aqueous phases (Charcosset and Fessi, 2005a). For example, a membrane contactor for the preparation of nanoparticles was described recently (Charcosset and Fessi, 2005a). The organic phase was pressed through the membrane pores via the filtrate side. The aqueous phase

circulated inside the membrane module, and swept away the nanoparticles forming at the pore outlets. It was shown that nanoparticles as small as 260 nm could be obtained with a 1000-Da nanofiltration membrane, a transmembrane pressure of 3 bar and a cross-flow rate of 1.7 m s 1. Very high fluxes were obtained with the 0.1 m pore size microfiltration membrane (1.6 m3/h m2), leading to the preparation of 1.8 10 3 m3 nanoparticles, with an average diameter of 360 nm, in 4 min. The advantages of this membrane contactor compared to other processes for nanoparticle preparation were demonstrated to be its scale-up ability and the possibility to control nanoparticle size by an appropriate choice of membrane. The potential disadvantage of direct membrane emulsification is the relatively low maximum disperse phase flux through the membrane (typically 0.01 to 0.1 m3/(m2 h)), needed to avoid the transition from a size-stable to continuous outflow zone and to avoid steric hindrance among droplets that may be formed simultaneously at the adjacent pores. To avoid this possible limitation, various operating methods were introduced such as rotating membranes (Zhu and Barrow, 2005) and repeated membrane extrusion of coarsely preemulsified feeds (Vladisavljevi et al., 2004). For example, Vladisavljevi prepared multiple w/o/w emulsions by multi-stage (repeated) premix emulsification using Shirasu-porous glass (SPG) membranes with a mean pore size of 10.7 m (Vladisavljevi et al., 2004). A coarse emulsion containing droplets with a mean particle size of about 100 m was homogenized 56 times through the same membrane at a constant pressure difference of 20300 kPa to achieve additional droplet homogenization and size reduction. However, membrane contactors will have to face competition from other emulsification and particle preparation processes, to find their place in industrial practice. 6. Conclusion Membranes have long been an integral part of biotechnology processes. The most well-known examples are ultrafiltration and microfiltration, which have become routine methods. The sterile filtration of fermentation media, purification of buffers and proteins are now standard practices. Other applications of membrane processes have been introduced more recently, as membrane bioreactors, membrane chromatography and membrane contactors. Although ultrafiltration and microfiltration are well-established processes, membrane bioreactors and membrane chromatography have certainly not obtained the expected levels of success. The reasons are related to a reticence of users for applying new technologies, and also to their

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C. Charcosset / Biotechnology Advances 24 (2006) 482492 Belanich M, Cummings B, Grob D, Klein J, O'Connor A, Yarosh D. Reduction of endotoxin in a protein mixture using strong anionexchange membrane absorption. Pharm Technol 1996:14250. Belfort G. Membranes and bioreactors: a technical challenge in biotechnology. Biotechnol Bioeng 1989;33:104766. Belfort G, Heath CA. New developments in membrane bioreactors. In: Crespo JG, Bddeker KW, editors. Membrane processes in separation and purification. NATO ASI Series E: Applied Science Dordrecht: Kluwer Academic Publishers; 1993. p. 12749. Belleville MP, Lozano P, Iborra JL, Rios GM. Preparation of hybrid membranes for enzymatic reaction. Sep Purif Technol 2001;25:22933. Brandt S, Goffe RA, Kessler SB, O'Connor JL, Zale SE. Membranebased affinity technology for commercial scale purifications. Biotechnology 1988;6:77982. Brites Alves AM, Moro A, Cardoso JP. Isolation of antibiotics from industrial fermentation broths using membrane technology. Desalination 2002;148:1816. Bueno SMA, Haupt K, Vijalayalakshmi MA. Separation of immunoglobulin G from human serum by pseudoaffinity chromatography using immobilized L-histidine in hollow fiber membranes. J Chromatogr, B, Biomed Sci Appl 1995;667:5767. Butterfield DA, Bhattacharyya D, Daunert S, Bachas L. Catalytic biofunctional membranes containing site-specifically immobilized enzyme arrays: a review. J Membr Sci 2001;181:2937. Cattoli F, Sarti GC. Separation of MBP fusion proteins through affinity membranes. Biotechnol Prog 2002;18:94-100. Charcosset C. Purification of proteins by membrane chromatography. J Chem Technol Biotechnol 1998;71:95-110. Charcosset C, Fessi H. Preparation of nanoparticles with a membrane contactor. J Membr Sci 2005a;266:11520. Charcosset C, Fessi H. Preparation of solid lipid nanoparticles with a membrane contactor. J Control Release 2005b;108:11220. Charcosset C, Limayem I, Fessi H. The membrane emulsification process a review. J Chem Biochem Technol 2004;79:20918. Charlton HR, Relton JM, Slater NKH. Characterization of a generic monoclonal antibody harvesting system for adsorption of DNA by depth filters and various membranes. Bioseparation 1999;8:28191. Chen GG, Luo GS, Xu JH, Wang JD. Membrane dispersion precipitation method to prepare nanoparticles. Powder Technol 2004;139:1805. Cheryan M, Mehaia MA. Membrane bioreactors. In: Mc Gregor, editor. Membrane separation in biotechnology. New York: Marcel Dekker; 1986. p. 255301. Chung T-P, Wu P-C, Juang R-S. Use of microporous hollow fibers for improved biodegradation of high-strength phenol solutions. J Membr Sci 2005;258:5563. Custer L, Physiological studies of hybridoma cultivation in hollow fiber bioreactors, PhD Thesis, University of California at Berkley, CA, 1988. Drioli E, Curcio E, Di Profio G. State of the art and recent progresses in membrane contactors. Trans IchemE 2005;83(A3):22333. Finger UB, Thommes J, Kinzelt D, Kula MR. Application of thiophilic membranes fort the purification of monoclonal antibodies from cell culture media. J Chromatogr, B, Biomed Sci Appl 1995;664:6978. Gavalas VG, Wang J, Bachas LG. Membranes for the development of biosensors. In: Bhattacharyya D, Butterfield DA, editors. New insights into membrane science and technology: polymeric and biofunctional membranes. Elsevier; 2003. p. 37992. Ghosh R. Protein separation using membrane chromatography: opportunities and challenges. J Chromatogr A 2002;952:1327.

potential advantages compared to other processes, such as classical reactors for biological reactions and conventional bead chromatography. In this sense, the examples of ultrafiltration and microfiltration may be advantageous, for which a continuous improvement in understanding the physicochemical phenomena governing the processes has been shown to enhance considerably the separation of proteins using their inherent electrostatic properties. In addition, the extensive study of hydrodynamics (i. e. Baruah et al., 2005; Kim and Zydney, 2005), combined with both numerical simulation and experimental investigation, has resulted in considerable improvement in performance of ultrafiltration and microfiltration, both in terms of fluxes and separation. New applications of membrane processes continue to emerge, such as membranes for the development of biosensors (Gavalas et al., 2003) and molecularly imprinted polymeric membranes for separation of molecules (Yoshikawa, 2002; Han et al., 2003). The very large range of chemical/physical mechanisms, which may occur in a membrane (e.g., permeation, reaction and mixing), appears to be one of the most attractive aspects of membrane processes. For this reason, there is no doubt that new successful applications will continue to appear in the near future. Their industrial success will again depend on their advantages compared to those offered by other competing processes and on their acceptance on the industrial market. Theoretical understanding of the physical and chemical phenomena governing these processes will also help to improve their performance and to facilitate their introduction in the biotechnology-based industries. References
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