CHL793 TERM PAPER

Application of Membrane Technology For Protein Purification

Submitted by : Vinita Kumari2011CH70188

Contents: Page no.1.Introduction 3-42. Effect of operating parameters on the separation of 4-8 proteins in aqueous solutions by ultrafiltration 3. Membrane fouling in UF and MF during protein separation 8-104. Modelling of constant flux based Protein Ultrafiltration 10-13 Notations: 14References: 15

IntroductionMembranes are used for separation and purification purpose in a wide range of industries including pharmaceutical ,food and dairy industry ,other chemical industries for waste stream treatment, water purification, defatting of skimmed milk and whey streams, bioseparation of fermentation products, deacidification of fruit juices etc. Membrane separation is based on the principle of selective diffusion driven by concentration (dialysis,osmosis) pressure (microfiltration ,nanofiltration, reverse osmosis, gas seperation, pervaporation) ,electric potential(electrodialysis) etc. There are a variety of membranes based on the material(ceramic,polymers,metals) from which they are made. Organic membranes are made of polyethylene, polysulfone, polyether sulfone, cellulose acetate etc and inorganic membranes made of aluminium oxide, zirconium oxide etc. There are liquid membranes also where thin layer of liquid film serves as a membrane. Membrane separation processes do not require additives and can be performed isothermally without much energy consumption and capital investment as compared to other thermal separation processes. The choice of membrane depends on the objective of the application for which it has to be used.Most protein based products need purification before their use. The requirements of protein purification are: Concentration enrichment Removal of specific impurities Protein stability enhancement Prevention of protein denaturationPurification of protein is challenging because complexity of their molecular structure, present in a very little amount in a solution(dilluted) before separation and also inherently unstable and subject to denaturation by heating, solvents and even shearing. Therefore conventional separation techniques such as distillation, solvent extraction absorption etc are of less use, mostly chromatography and membrane separation processes are used. Chromatography has some limitations-difficult to scale up, expensive to operate, batch operation and detail knowledge of solution system.Although all membrane separation processes are used for protein separation or purification, the most widely used are pressure driven membrane separation processes ultrafiltration(UF), microfiltration(MF)and nanofiltration(NF). MF membranes are especially applicable for the separation of fine particles in the size range of 0.110.0 m. While UF membranes having pore size in the range of1100 nm are designed to provide high retention of proteins and other macromolecules . The applications of UF are limited to systems where the solutes to be separated have more than 10-fold difference in molecular weight (MW) in comparison with protein molecules. Molecular size becomes the sole criteria for separation purposes in such cases. However, it is possible to separate solutes having comparable molecular weight by adequate manipulation of the parameters such as pH, ionic strength, and transmembrane pressure (TMP).Ionic strength should be kept low so that the thickness of diffused double layer of charged solute is significant leading to high retentate while uncharged solute permeating the membrane. Operating pH should be near to isoelectric point(pI) of transmitted protein and far from isoelectric point membrane of retained protein.

RecycleBuffer Condition

Feed

TMP

Membrane unit

Schematic for an ultrafiltration processPermeateNF (Nanofiltration) is useful for separation of peptides due to the suitable cut-off of the NF membranes and due to the electrochemical effects, which plays an important role in the case of charged molecules. Negatively charged membranes have been applied to get cationic peptides (having antibacterial properties) from cheese whey. A study on the desalting of peptide fractions from whey protein hydrolysate using NF membranes has proved the occurrence of specific rejection phenomena involving negatively charged peptides by NF membranes. Variation in pH and the ionic strength of the hydrolysate phase has proved the charge effects on performance of peptides separationFactors affecting the performance of an protein separation/purifiacation are: Ionic Strength of solution pH of solution transmembrane pressure Stirring speed(if mixing required) Membrane charge type(positive or negative) Membrane material Initial Protein ConcentrationLast three factors cannot be modified and are varied prior to conducting experiment.

Effect of operating parameters on the separation of proteins in aqueous solutions by ultrafiltration:(BSA (Bovine Serum Albumin))

Effects of TMP, initial concentration, and pH on the rejection of protein:R =1 Cp/CfCp: protein concentration in permeate Cf: protein concentration in feedIt has been found that rejection of protein increases with pH and increasing TMP and initial concentration. At low TMP a maximum rejection of protein is found at low pH, particular at pH lower than pI(4.9 for BSA) value of the protein . This is because the positively charged protein would adsorb onto the negatively charged membrane at pH < pI. However, the influence of pH is negligible at high TMP and high initial concentration

ConcpressureRejection

RejectionmpHpressure

Effect of pH and TMP on the flux of protein solution:It has been observed that flux increases sharply with TMP and thenlevels off at lower initial protein concentration. Effect of solution pH is more pronounced at low protein concentration. At low pH(6 for BSA) positively charged molecules would absorb easily on the negatively charged membrane and block the membrane pores. However the repulsive force between negatively charged protein and membrane reduce the fouling at highr pH(>7).

Effect of pH and P on UF flux

Effect of pH and TMP on the separation factor=1-RBSA/1-RHbRBSA=rejection of BSA moleculesRHb= rejection of Hb moleculesHigher separation factor is obtained at lower TMP and pH near to the pI (isoelectric point) of Hb because of the self aggregation of the neutral Hb molecules(pH7.1) and they are not adsorbed on the surface of membrane by electrostatic attraction resulting in reduced fouling.At this pH BSA molecules can easily pass through the pores of membrane.At pH 6 or below serious fouling is observed becaue of the preferential adsorption of positively charged Hb molecules on surface of negaitively charged membrane.At pH greater than 7.5 separation factor is less than 1 which indicates that amount of Hb passing through the membrane is greater than the amount of BSA molecules(RHb> RBSA).It occurs due to greater repulsion between BSA molecules and membrane as compared tothat between Hb molecules and membrane.

C0BSA=500ppm

C0BSA=100ppm

Membrane fouling in UF and MF during protein separation:Membrane fouling refers to the irreversible alteration in membrane properties, caused by specific interactions between feed stream components and membrane.Fouling of membrane during practical application for protein separation results from its adsorption on membrane surface which increases hydraulic resistance to flow, reduced filtration flux rate an adverse effect on efficiency and economics of protein recovery processes.Proteins are difficult foulants to deal with because they readily adsorb onto membrane surfaces and pore walls. This leads to the formation of a secondary barrier that decreases permeate flux and solute selectivity is altered. Therefore, to minimize fouling by making the membrane surface hydrophilic is a challenge for achieving better membrane performanceFouling can occur in following ways: The formation of a gel layer as a result of concentration polarization Adsorption of specific species on the membrane surface and inside the pore structure Deposition and pore blockage after the formation of protein aggregates caused by protein denaturation.The techniques which are used to characterize membrane fouling include measurements of the flux decline at constant pressure, and the pressure increase during constant flow rate permeation.

Reducing Fouling: Introduction of microsieves having well structured morphology and controlled porosity,in place of conventional MF membranes. It results in good separation behavior and enhanced flow rate. It allows low pressure driven operation and thus reduced operational cost. It helps in reducing fouling because of the smooth surfaces and hindering the trapping of proteins inside the pore network which normally occurs in polymeric membranes. Protein fouling during UF is mainly due to the formation of a secondary (gel) layer on the upper surface of the membrane which provides an additional resistance to both, solute and solvent transport across the membrane.Hence surface modification by increase in membrane surface hydrophilicity can effectively minimize protein adsorption and prevent membrane fouling. These methods include coating, surface graft polymerization and chemical modification to reduce the UF membrane fouling during protein separation.

Schematic diagram for mechanism of membrane fouling during protein seaparation

Modelling of constant flux based Protein Ultrafiltration:A simple mathematical model for increase in transmembrane pressure during constant flux ultrafiltration considering the contribution of concentration polarization, rapid initial fouling and long term fouling has been discussed below.In constant pressure ultrafiltration flux decreases continuously and hence it may be concluded that osmotic pressure and rate of fouling would decrease. The model assumes that a constant pressure ultrafiltration process is made of large number of very small constant flux steps. It has been assumed that initial flux in constant pressure ultrafiltration would correspond to pure water(or buffer) flux of a fresh membrane at the operating pressure. Therefore the first constant flux step in the model represent this. In all subsequent step osmotic pressure at that instant and cumulative resistance increase due to membrane fouling has been taken into account to calculate the permeate flux. The protein used for the experiment is HAS(human serum albumin) and the membrane is polyethersulfone(PES).Concentration polarization builds up in a matter of minutes while fouling may take place during the entire duration of an UF process. In constant pressure UF, the effects of fouling and concentration polarization are observed in the form of decline in permeate flux with time.

Concentration polarization and rapid foulingBuffer filtration throughfouled membraneBuffer filtration through fresh membrane

TMP

Long term linear fouling (slope=Jv)(

Time

Approach for predicting flux decline in constant pressure ultrafiltration.

Equations governing the model:Increase in TMP with time explained by modified form of osmotic pressure resistance model: P = + Jv(R0m + Rm* + t) Rf changes with time due to deposition and adsorption of foulant and expressed as: Rf = Rm+ t (=m/Jv)The mass balance equation for protein(foulant) for the concentration polarization layer assuming that diffusion and convection within this layer takes place perpendicular to membrane surface: Permeate flux can(Jv) be expressed using the osmotic pressure model: Jv=(P-)/Rm Jv,i =( P i1)/Rmi1 + ((Rm* t)/tR) + ((mi1 *t)/Jv,i1) ttRInitial condition:C(y,0)=Cb at t=0Boundary Conditions:For y=0 C(0,t)=Cb , For y= JvC(,t)=D

Detailed scheme for predicting permeate flux decline including working equations.

Notations:C: foulant(protein) concentration (kg/m3)Cb:foulant bulk concentration (kg/m3)Cw:foulant wall concentration (kg/m3)D:diffusion coefficient of foulant (m2/s)Jv:volumetric permeate flux (m/s):water flux (m/s)k::mass transfer coefficient (m/s)m:slope of the linear portion of TMPtime profile in constant flux ultrafiltration (kPa/s)P:transmembrane pressure (kPa)Rf: fouling resistance (kPas/m)Rm :total membrane resistance (kPas/m)R0m :membrane hydraulic resistance (kPas/m)Rm*:initial rapid fouling constant (kPas/m)t:time (s)t:small time increment (s)tR:duration of initial rapid fouling phase (s)y:distance from the edge of concentration polarization layer toward membrane (m):fouling rate constant (kPa/m):boundary layer thickness (m):osmotic pressure (kPa)

References:[1]Almcija etal., 2007 M.C. Almcija, R. Ibez, A. Guadix, E.M. Guadix Effect of pH on the fractionation of whey proteins with a ceramic ultrafiltration membrane,Journal of Membrane Science, 288 (2007), pp. 2835[2] R.W. Baker,Membrane technology and applications,Wiley, Chichester (2004)[3]M.C. Almcija, R. Ibez, A. Guadix, E.M. Guadix,Effect of pH on the fractionation of whey proteins with a ceramic ultrafiltration membrane,Journal of Membrane Science, 288 (2007), pp. 2835[4]M.Y. Teng, S.H. Lin, C.Y. Wu, R.S. Juang Factors affecting selective rejection of proteins within a binary mixture during cross-flow ultrafiltration,J. Membr. Sci., 281 (2006), pp. 103110[5]W.R. Bowen, D.T. Hughes,Properties of microfiltration membranes. Part 2. Adsorption of bovine serum albumin at aluminum oxide membranes,J. Membr. Sci., 51 (1990), p. 189[6] R.F. Boyd, A.L. Zydney,Analysis of protein fouling during ultrafiltration using a two-layer membrane model,Biotechnol. Bioeng., 59 (1998), p. 451[7]R. Ghosh. Study of membrane fouling by BSA using pulsed injection techniqueJ. Membr. Sci., 195 (2002), p. 115[8]E. Matthiasson The role of macromolecular adsorption in fouling of ultrafiltration membranesJ. Membr. Sci., 16 (1983), p. 23