Membrane-basedMembrane-based techniques techniques for the separation for the separation and purification ofand purification of P Proteinroteins swww.378700000.com Membrane-based techniques for the separation and purification of proteins1Introduction2Pressure-driven membrane technologies 3Membrane chromatography4Electrophoretic membrane contactorwww,378700000.comIntegrated membrane technologies5CONTENTS6Conclusions1. IntroductionDefinitionAdvantageApplicationA membrane can be described as an interphase,usually heterogeneous, acting as a barrier to the flowofmolecular and ionic species present in the liquids and/or vapors contacting the two surfaces.Advantage:(1)selective transport,(2)efficientseparation,(3)not require additives,(4) less energy consumption.Application:(1)demineralization,(2) desalination/purification of water,(3)bioseparation of fermentation products(4)milk fractionation,(5)decaidification of fruit juices.Type:(1) microfiltration (MF),(2) clarification, sterile filtration and ultrafiltration (UF) ,(3) Nanofiltration (NF),(4) High-performance tangential flowfiltration (HPTFF).www,378700000.comTypeDefinitionMF membraneswere tailored to retain cells and cell debriswhile allowing proteins and smaller Molecules to pass into filtrate.UF membranes were designed to provide high retention of proteins and other macromolecules.(NF) was defined as a process that separates solvent, monovalent salts, small organics from divalent ions and larger speciesHPTFF is a two-dimensional purification method that exploits differences in size and charge characteristics of protein/biomolecules.www,378700000.comwww,378700000.com Disadvantage:(a)limitation of the process stream for relatively low conductivity of feed stream, (b) a high-energy requirement, (c) substantial heat production, and (d) changes in the process feed due to reaction at the electrode PES is widely used UF membrane material, because of its high rigidity, creep resistance, good thermal and dimensional stabilities. Other types of polymeric UF membranes such as polyacrylonitrile membrane, regenerated cellulose membrane, cellulose acetate membrane and ceramic membranes.www,378700000.com2.Pressure-driven membrane technologies for separation/purification of proteins2.1.Proteins separation/ purification by MF2.2.rotein separation by UF2.3.Advanced UF techniques for proteins separations2.4.Protein separation/purification by NFwww,378700000.com2.5.Membrane fouling during protein separation by UF and MF2.1. Proteins separation/purification by MFModule configuration of MF include hollow-fiber, tubular,flat plate, spiral-wound and rotating devices. The two standardmodes of operation are dead-end and cross-flow configurations are shown in Fig. 2. These devices show significant increases in protein transmission and capacity.MF is widely used for the separation, purification and clarifying of protein-containing solutions, e.g. for the recovery of extracellular proteins produced via fermentation and for the removal of bacteria and viruses in the final formulation of therapeutic proteins.www,378700000.com2.1.1. Advanced MF under electric fieldMuch effort is still being devoted to developing new membrane modules with improved mass-transfer characteristics for UF and MF processes. Electrically enhanced membrane filtration (EMF) is an advanced technique, which consists in superimposing an electrical field to a conventional embrane filtration unit. In EMF, the electrical field acts as an additional driving force to the transmembrane pressure.Advangtage:(1) selectivity enhancement,(2) improve protein solutions permeation flux,(3)reduce the surface layer of a membranewww,378700000.com2.2. Protein separation by UFUF membranes, based on variety of synthetic polymers, have high thermal stability, chemical resistivity, and restricted the use of fairly harsh cleaning chemicals.Modules:(1) Hollow fiber,(2) flat-sheet cassettes,(3) spiral-wound cartridges, (4) tubular modules,(5) enhanced masstransfer devices www,378700000.comProtein fractionation is rapidly becoming more selective through improvements in membrane and module design.www,378700000.com2.3.1. Protein separation using charged UF membranesCharged UF membrane separation process involved both size and charge based exclusion rather than simply size-based separation of protein molecules, as in the case of UF.Factor:(1) pH values ,(2)ionic strengths,(3) permeate flux, (4)and system hydrodynamics.Advantange: extremely low fouling due to electrostatic repulsion.www,378700000.comMaterial:(1)polyethersulfone, (2)polysulfone, (2)cellulose acetate, (3)regenerated cellulose,(4) poly (ethylene glycol) (PEG) ,(5) poly (furfural alcohol) (PFA).2.3.2. UF in the presence of electric field (electro-ultrafiltration) The use of electric field in UF goes back to the first study carried out by Bechhold by imposing electric field in UF and utilized a combination of electroosmosis and electrophoresis to purify colloids in an apparatus he called an electro-ultrafiltration (EUF). EUF is an effective method to decrease gel layer formation on the membrane surface and to increase the filtration flux, owing to electrokinetic phenomena such as electrophoresis and electroosmosisFactor:(1) electric field,(2) solvent flowwww,378700000.comAdvantage:(1)enhance the filtration rate ,(2)increases the separation efficiency of the UF of proteins .For this reason, attention has been directed to the use of pulsed electric fields.Advantage:(1) less energy,(2) higher flux ,(3) reducing fouling,(4) restoring high permeation rate.www,378700000.comDisadvantage:(1) limitation of the process stream for relatively low conductivity of feed stream, (2) a high-energy requirement, (3) substantial heat production, and (4) changes in the process feed due to reaction at the electrode.The application of the electric pulse in the cleaning membrane surface was an effective means in reducing fouling and restoring high permeation rate.2.3.3 UF in the presence of ultrasonic fieldconcentration polarization and membrane fouling lead to the declination of permeates flux.concentration polarization reason:a concentration gradient of the retained components is formed on or near the membrane surface.Ultrasound generates acoustic streaming and cavitation bubbles in a liquid medium, Cavitation bubble causes microstreaming,microstreamers,microjets, and shock waves.membrane fouling reason: accumulation of proteins drawn toward filtering surface by convective flow of filtrate through the membrane.Various methods have been used to reduce the negative effects of concentration polarization and fouling for enhance the permeate flux and membrane separation efficiency such as Ultrasonic physical effects and sonochemical effects.www,378700000.comRelease from a particle-fouled surface mechanismHigher frequency ultrasound tends to have higher energy absorption and thus greater acoustic streaming flow rates than lower frequencies for the same power intensity, higher power intensities lead to greater acoustic streaming flow rates.This mechanism causes bulk water movement toward and away from the membrane cake layer, with velocity gradients near the cake layer that may scour particles from the surface.The effect of ultrasonic wave onseparation performance of protein mixture by UFThe effect of ultrasound on the flux and solute rejection in cross-flowUF of BSA-lysozyme binary protein mixture using PES membrane ultrasonic wave not only enhanced the UF flux but also increased the lysozyme rejection.ultrasound wave (25 kHz and 240 W) resulted in an increase of UF flux by 135% and 120% with PES membrane at pH: 11 in the upward and downward modes, respectively, in contrast to the case of without any ultrasound.Muralidhara and Tarleton:electric and ultrasonic fields can reduce membrane fouling and in turn of enhanced flux.additionWakeman and Williams:Both electrical and ultrasonic fields reduced the fouling when applied individually, but the extent of improvement by the ultrasonic field could be minimal.Notice: the effectiveness depends on many factors, such as orientation and position of ultrasonic field, ultrasonic frequency and power, ultrasonic radiation angle, position of ultrasonic vibration platein the membrane module, membrane material, membran housing,operating pressure and the fouling material.Reusit: ultrasonic cavitation, acoustic streaming, ultrasonicinduced vibration of membrane and ultrasonic heating was the main causes.2.3.4. High-performance tangential flow filtrationHPTFF : an emerging technology 、similar size（Conventional ten-fold in size，now less than three fold in size）.Optimum selectivity and throughput，enhanced through module design and process configurations.Sieving behavior impact:Optimizations of buffer pH and ionic strength.A twodimensional unit operation,protein concentration, purification and buffer exchange can be accomplished in a single unit operation.www,378700000.comSome strategies to achieve highresolution separations(1) proper choice of pH and ionic strength to maximize differences in the hydrodynamic volume of the product and impurity.(2) use of electrically charged membranes to enhance the retention of like charged proteins.(3) operation in the pressure-dependent regime to maximize the selectivity.(4) use of a dia-filtration mode towash impurities through the membrane.www,378700000.comComparison of flowand pressure profiles forconventional TFF module and co-flow arrangementThis system overcomes some of the problems associated with conventional MF/UF configurations and equipment and is reported to give high degree of protein fractionation.www,378700000.com2.4. Protein separation/purification by NFNF：suitable cut-off of the NF membranes and the electrochemical effects.1、Negatively charged membranes have been applied to enrich cationic peptides with antibacterial properties from cheese whey.2、Pouliot：the separation of peptides from tryptic hydrolysates of whey proteins with charged UF/NF membranes.3 、Variation in pH and the ionic strength.www,378700000.com2.5. Membrane fouling during protein separation by UF and MFMembrane fouling：adsorption on membrane surface significantly increases hydraulic resistance to flow, reduced filtration flux rate and induced unfavorable effect on efficiency and economics of protein recovery processes.Fouling forms：（i）The formation of a gel layer due to concentration polarization.(ii) Adsorption of species on the membrane surface and inside the pore structure.(iii) Deposition and pore blocking after the formation of protein aggregates due to denaturation.www,378700000.comHo and Zydney： developed a combined membrane fouling modelpore blockage and cake filtration to describe flux decline.The model shows a smooth transition from pore blockage to cake filtration, and is in good agreement with flux decline data obtained during bovine serum albumin filtration using polycarbonate track etched membranes.But internal fouling was completely neglected.welcome to use these powerpoint templates, New Content design, 10 years experiencewww,378700000.comMicrosieves,the advantage is the larger permeate flux, which al lows low-pressure operation and savings in the operational costs and another advantage of microsieves over MF membranes is their structural design, a very thin selective layer and perfectly shaped straight pores.Surface modification ,such as coating ,surface graft polymerization, and chemical modification.Nakao: studied that protein fouling during UF was entirely due to the formation of asecondary (gel) layer on the upper surface of the membrane.Jiang. synthesized pegylated PES via a reaction of sulfonated PES with oligomeric poly (ethylene glycol) (PEG). The modified membranes showed superior resistance to BSA adsorption in compare with unmodified counterparts.www,378700000.com3. Membrane chromatographyTITLEAffinitychromatography has proven its ability, efficiency and time stability for high-resolution separation and analysis of protein.Three basic requirements:1. Abiospecific ligand must be available for target molecule to be separated or purified.2. The ligand must have reactive chemical groups for its covalent attachment to a chromatographicmatrix.3. The membrane matrix should be easily derivatised (functional group should be easily available for the covalent attachment of one of the components of the binding pair).www,378700000.comThe selection of the ligand is influenced by two factors: the ligand must exhibit specific and reversible binding affinity for the target substance(s) and it must have chemically modifiable groups that allowit to be attached to the matrix without destroying binding activity.ligand molecules are immobilized on the porous surface of the embedded particles and the mixture containing the protein of interest is passed through the affinity membrane.welcome to use these powerpoint templates, New Content design, 10 years experiencewww,378700000.comThree types of membrane adsorbers :flat sheet, hollow fiber and radial flowFlat-sheet membrane adsorbers, the liquid was usually introduced to themembrane surface. Stacks of several flat sheets were housed within membrane modules.A hollow-fiber membrane adsorber usually consists of a bundle of several hundred fibers potted together within a module in a shell and tube heat-exchanger-type configuration.Radial flow adsorbers were claimed to be suitable for large-scale applications.welcome to use these powerpoint templates, New Content design, 10 years experiencewww,378700000.com3.1. Microporous materials for membrane chromatographyMicroporous or macroporous membranes.Membranes are commonly made of natural or synthetic polymer base materials include cellulose, aliphatic polyamides , aromatic copolymers ,hydrocarbon polymers ,polyvinyl alcohol, glass hollow fiber, synthetic copolymer, etc.Materials can be modified by chemical activation, coating and grafting.www,378700000.com3.2. Affinity ion-exchange materials for membrane chromatographyModern ion-exchange materials are prepared from synthetic polymers such as styrene divinylbenzene copolymers.Li. have used a cationexchange monolith, as chromatographic supports for separation of four standard proteins.Saiful. embedded ion-exchange resins in EVAL (a random copolymer of ethylene and vinyl alcohol) membranes for enzyme recovery.www,378700000.comIon-exchange membranes materials can be produced either by modification of commercially available MF membranes or by embedding of IEX-resins into a polymeric porous matrix. 3.3. Application of membrane chromatography for protein separationThe uses of ion-exchange and affinity interactions are more widely reported, but only small work has been done on hydrophobic interaction and reversed-phase based membrane chromatography of proteins.The ligands（配体）used for affinity-membrane chromatography can be broadly classified into four types: iimmunoaffinity ligands,iiprotein, iiilow-molecular-mass ligands, ivother ligands.Lysozyme（溶菌酶） separation from egg white was achieved efficiently using macroporous chitin membrane,with98% purity.www,378700000.comWheat germ agglutinin（麦胚凝集素） (WGA) is an important and very expensive lectin（外源凝集素） useful. Macroporous chitin membranes with large pore sizes (average 18 m) and high adsorption surface were used. A two-step elution（洗脱） was employed. A purification factor of 5.5 and an activity yield of 40% were obtained.About 25 mg of pure wheat germ agglutinin was obtained from 50 g.One of the major limitations of is nonuniform flow distribution（非均匀流体分配） across the membrane, due to the large diameter-to-length ratio of the modules.www,378700000.comApplications Thiophilic membranes（嗜硫膜） for the purification of monoclonal antibodiesHollow-fiber membranes（中空纤维膜） for the separation of IGStrong anion-exchange membranes for reduction of endotoxinIon-exchange membranes for the isolation of antibacterial peptides from lactoferrinCation-exchange membranes for the purification of alpha viruses Affinity membranes for the separation of MBP fusion proteinswww,378700000.com4.Electrophoretic membrane contactor for the separation of proteinsDifferent studies were devoted to find out the operating modes for scale-up of electrophoretic separations. One of them is continuous flow electrophoresis(CFE)（连续流动电泳）.The limitations were in terms of production capacity or productivity, demonstrated and it had a strong relationship between resolution and productivity. It was found that the productivity could not be increased over a certain limit,typically about few milligrams per hour.www,378700000.comTo overcome these limitations, electro-membrane operations offered the possibility to increase the productivity without compromising from separation efficiency. The most common electromembrane operation is electrodialysis (ED)（电渗析） in which ion-exchange membranes are used.In that case, the porous membrane acts as a contactor and the separation is achieved with respect to the difference between the mass flow rates of the species.Electrophoretic membrane contactor was developed for the separation of binary mixture of proteins by using ion-exchange and ultrafilter membranes.www,378700000.comwww,378700000.com5.Integrated membrane technologies for protein separationA combination of different membrane processes gives interesting benefits, which cannot be achieve by single membrane operation. The possibility of redesigning overall industrial production by the integration of various already developed membrane operations is becoming of particular interest.The largest membrane area was installed in the dairy industry. It has been estimated 2,000,000 m2 membrane area were installed for the fractionation of milk and whey.www,378700000.comMembrane process for food and dairy industryMilk and dairy industryThe integration of membranes has been implemented throughout the milk and dairy processing chains-milk reception,cheese making, whey protein concentration, fractionation of protein hydrolysates, waste stream purification and effluents recycling and Treatment. Food and beverage industriesRecent industrial applications have been developed for fruit, vegetable and sugar juices and beverages (vegetable proteins, beer, and wine). Further integration of membrane operations were designed in such a way that at each processingstep, end products, co-products and wastes are given even attention.www,378700000.comMembrane process for food and dairy industrywww,378700000.comMembrane processRemoval of bacteria and spores from skim milk (cold pasteurization)MF reduces the amount of bacteria, spores without affecting the taste of milk, and provides longer shelf life than pasteurization.Besides the production of consumption milk with extended shelf life（保质期）,this method can be used as pretreatment of skim milk（脱脂牛奶） for the production of raw milk cheeses and the reduction of spores in acid cheese milk.A recent development is the microsieves, which was made with micro-machining technology.With microsieves, a high reduction of bacteria can be achieved at low transmembrane pressure, since these membranes have very high permeabilities.Hence, the use of microsieves seems very promising.www,378700000.com Recovery of serum proteins and cheese production During cheese production, the milk is coagulated by precipitation of the milk proteins.Polymeric ultrafilter membranes are fully retentive for whey protein to remove lactose and minerals. Whey（乳浆） contains most of the dissolved salts and sugars present in the original milk and about 25%of the original protein. Initially whey was discharged to the sewer because its high salt and lactose content makes direct use as a food supplement difficult. www,378700000.comThe objective of the UF step is to concentrate the protein as much as possible to minimize evaporator-drying cost and simultaneously remove the lactose（乳糖）.www,378700000.comRecently, cross-flow MF has been used to separate cells in continuous fermentation processes for a successful lactic acid recovery approach.Recovery of heterogeneous immunoglobulin (IgG) from transgenic goat milk by MF.Recovery of naturally glycosylated therapeutic proteins produced from animal cell cultures by MF.Recovery and purification of yeast alcohol dehydrogenease (ADH) from bakers yeast for the extraction of an intracellular enzyme product.Biotechnology industrywww,378700000.com Since long it has been an integral part of biotechnology processes, the well known examples are MF and UF, which have become routine methods for protein separation/ fractionation. The development of membrane chromatography, HPTFF and electrophoretic membrane contactor enable for the complete purification/separation of proteins using membrane systems.Future trends of membranes in biotechnology will be driven by higher selectivity, lower cost of production, and enhanced membrane throughput.New applications :membrane biosensors and molecularly imprinted polymeric membraneswww,378700000.com6. Conclusions