Understanding flux patterns in membrane processing of protein solutions and suspensions

451

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Understanding flux patterns in membrane processing of protein

solutions and suspensions

Membrane mlcrofiltratlon is one of the most effective ways of separating proteins

from materials of larger size, such as microbial cells and cell debris. Ultraflltration

is the most effective means of concentrating protein solutions. ThB review

describes the scope and operating patterns of these processes. A major goal for

membrane technologists is the development of predictive descriptions of

membrane processes, based on a rigorous understanding of the underlying

physico-chemical phenomena. The emphasB ~s placed on quantitative analys~s of

the interracial aspects and mlcrohydrodynamics of membrane processes. From

existing knowledge, it is already possible to define a number of guldehnes for

successful filtration process operation.

Richard Bowen

Membrane separation technology has evolved from a small-scale laboratory techmque to a large-scale industrial process during the past 30 years. At the present time, a discussion among process-design engineers of separation problems for fluid systems will almost inevitably lead to the question off whether the prob- lem under discussion can be solved by a membrane process ]. The apphcatlon o£ membrane processes to separation problems in flmd systems has been especially swift in some b]otechnological industries 2 due to the equipment investment opportumt]es aris- ing In this relativcly new and rapidly growing industrial sector.

In simple terms, membranes may be thought of as h~gh-technology filters. A classification of" the main types of membrane processes Is given in Table 1. It may be seen that the processes described allow the separanon of materials over a size range covenng four orders of magnitude, fiom 10Fro to I nm. This range of"sizes corresponds very well to the sizes of 'many o£ the materials that have to be processed in the b~otech- nology industry.

This article is concerned with the two membrane processes that are most w~dely used in bmtechnology - microfiltranon and ultrafiltration. Some of-the ways m which these techniques are used in the processing of"protein solunons are illustrated in the recovery process outlined m Fig. 1. Such a recovery process rmght be used, for example, in the large-scale productmn of extracellular enzymes.

R. Bou,en i~ at the Blodlemical En£meerin£ Gtoup, Depart, lent of Chemical tgn£meerin~, Umver~d 7 Co/le w o/ Su,ansea, University el-l, Vales, Su,ansea, UK Gq2 8PP,

Table 1. Classification of the major types of membrane processes

Applications/ examples of

Separation materials Process Driving force range separated

Microfiltratlon Pressure gradient 10-0.Item Small particles, large colloids, microbial cells

Ultrafiltratlon Pressure gradient 0.11.m-5nm Macromolecules, emulsions

Reverse Pressure gradient <Snm Desahnat~on, osmosB small organics

Electrodialysis Electric-field <5rim Desahnation, gradient small organics

Electroflltrat~on Combined pressure 10 Fm-5 nm and electrm-field gradient

As for microfiltration and ultraflltration

D~alysB Concentrat=on <5 nm Treatment of gradient renal failure

Pervaporation Partial pressure Volatile Organic matenats m~xtures,

water-organ=c mixtures

Gas Pressure gradient Gas Gas mixtures permeation molecules

© 1993, Elsev, er Science Publishers Ltd (UK) TIBTECH NOVEMBER 1993 (VOL t l )

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Wash water

From fermenter

, Stirred tank

~ Microflltration ]

Storage tank

Multi-stage ultrafiltration

Storage tank

Ultrafiltrat~on

Blend and ] standardize

Microfiltration

Enzyme solution

Biomass

Permeate

.~ Permeate

Salt

Figure 1 Flow dtagram of the recovery of an extracellular enzyme using micro- and ultraflltratlon.

In the example shown m Fig. 1, mlcrofiltration is used to remove the biomass at the end of the fermen- tation. As (in this case) the enzyme has been secreted into the extracellular medium, the microfiltration membrane retains the biomass, allowing the enzyme to pass through. A great advantage o f membrane microfiltration over the compet ing techniques o f centrifu- gatlon and conventional filtration is that separation o f the blomass from the extracellular m e d m m is essentially 100%. Microfiltration is also used in the final process o f the schemc described in Fig. 1 to polish and sterdize the product.

P, efcrrmg again to the scheme dlustrated in Fig. 1, ultrafiltranon is used to concentrate the enzyme solutions. The concentrat ion steps are carried out in a number o f stages to opumize the process economics. For each of the initial rounds o f ultrafiltratlon, concentration o f the enzyme by a factor o f five would typically be achieved, with concentration by a factor of ' ten being achieved in the final stagc.

In the product ion o f lntracellular proteins, m e m - brane microfittration can be effecnvely employed to separate cell fragments following disruption. M e m - branes are also widely used to concentrate proteins m

the food industry, especially the dairy industry. M e n > brane ultrafiltration can also be used to fractionate proteins; for example, albumins and globulins, although very careful process control is required, even if the proteins differ substantially in size.

The nature of synthetic membranes and membrane modules

An understanding o f the operation o f membrane processes requtres some knowledge o f the nature of the membranes themselves 3. The membranes used for mIcrofiltratIon and ultrafiltratlon are niost commonly made o f polymeric materials. Initially, most m e m - branes were cellulosic, although cellulose is n o w being replaced by polyamide, polysulphone, polycarbonatc and a number o f advanced polymers 3. These synthetic polymers have improved chemical stabdity and better resistance to microbial degradation, making them very suitable for use In filtration membranes.

Most microfiltrauon membranes have a symmetrical pore structure, with porosities as high as 80%. These membranes arc typically 50 -100 bun thick. Ultrafil- tration membrancs have an asymmetric pore structure comprising a 1-2 btm thick top layer o f the finest pore size, supported by a ~100 Ixm thick matrix that IS more openly porous. An asymmetric structure is essential if reasonable mcmbrane-permeat ion rates are to be achieved with membranes o f such a small pore size. Another important type ofultrafiltratlon membrane is thc thin-film composite membrane, which comprises an extremely thm layer, typically -1 btm thick, o f the finest pore structure, deposited on a more openly porous mamx. The thin layer and the support are made from different materials.

Microfiltration membranes are typically specified m terms o f their pore &ameter, whereas ultrafiltratlon membranes are usually specified m terms o f their 'molecular weight cu t -o f f ' (MWCO) . This value m&- cates that 95% o f molecules o f the specified molecular weight would bc retained by the nrembrane. Both types ofspccif icanon need to be used with caution as they depend on the means o f measurement, which vanes fi-om lnanufacturer to manufacturer.

A recent significant advance has been thc development ofm~crofiltranon and ultrafiltratlon membranes composed o f inorgamc oxide materials. Alumina (A1203) and zirconia (ZrO2) arc the materials most c o m m o n l y used. The main advantages o f morgamc membranes compared with polymeric types are their greater stability at high temperatures, which facd~tates steam sterilizauon, possibly a narrower pore-size distribution and increased resistance to fouhng.

Po lymenc membranes are produced as flat sheets, m the form of hollow fibres o f &ameter 0 .1-2 .0ram, or as tubes o f diameter 10-25 mm. Ceramic membranes arc produced as tubes (sometimes multiple tubes in a matrix), or as metal-mesh-supported composites. Membranes for industrial-scale operations are supplied in the form o f modules, which give membrane areas In the range 1 - 2 0 m 2. The four most c o m m o n types o f modules are"

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(1) Tubular, which 1s widely used when it is advan- tageous to have conditions o f turbulent flow. (2) Flat sheet, an arrangement that is similar, m many ways, to a convenuonal filter press. (3) Spiral wound, comprising several fiat membranes separated by turbulence-promot ing mesh separators and formed into a 'Swiss roll'. (4) Hollow fibres, consisting o f bundles o f fibres sealed m a tube with, for biotechnological apphcanons, the feed passing d o w n through the centre o f the fibre, gwmg controlled laminar flow.

'Dead end' membrane mlcrofiltration, in which the particle-containing fluid is pumped directly through a polymeric membrane is used in the mdustrial clarifi- canon ofhquids. Such a process allows the separanon of particles down to 0.1 I~m or less, but is oflly suitable for feeds containing very low concentrations o f particles, as otherwise the membrane becomes clogged too rapidly. Most ultrafiltrauon and mlcrofiltration processes in biotecbnology are operated under 'cross- flow' as shown in Fig. 2a. The feed soluuon is pumped parallel to the membrane at a velocity in the range o f 1 -8ms 1 with a pressure difference o f 0 . 1 - 0 . 5 M P a ( M N m 2) across the membrane. Llqmd permeates through the membrane and feed emerges in a more concentrated form on exit from the module.

The advantages o f cross-flow membrane separanons are:

(1) A higher overall liquid removal rate is achieved by preventing the formauon o f extensive deposits on the membrane surface. (2) The concentrate (retentate) remams in a mobile form suitable for further processing. (3) The solute content o f the concentrate may be vaned over a wide range. (4) It may be possible to ffactionate solutes o f &fferent sizes.

Membrane modules are configured in series aim in parallel m various ways to produce a plant with the required separation capability. T w o o f the possibilities are illustrated in Figs 2b and c. The arrangement illustrated in Fig. 2b is hkely to be used for small-scale batch processing or development rigs. In essence, it is a basic pump-rec i rculauon loop. The process feed is concentrated by pumping it from the tank and across the membrane module at an appropriate velocity. The partially concentrated retentate is recycled into the tank for further processing, while the permeate is stored or &scarded as required. Product washing is frequently necessary, and this is achieved by a process known as diafiltrauon in which wash water is added to the tank at a rate equal to the permeation rate. Fig- ure 2c depicts a 'feed and bleed' system, which is well- suited for larger-scale biotechnologlcal operauons. The imtiat sequence is similar to a batch system, In that the retentate is totally recycled at first. However , when the required final solute concentrat ion is reached within a loop, a fraction o f the loop contents are bled

a Permeate Membrane

Process feed cross-flow ~

Permeate

........... Retentate

b To drain or store tank Permeate Compressed air

. . . . . . I c><>F>~ - for backflush

woo~ I~,,,,ia I Concentrate

~ microfiltration L... ~ t module I

Variable speed Heat Flowmeter pump exchanger

C Permeate Permeate Permeate Feed

Retentate

Stage 1 Stage 2 Stage 3

Figure 2 (a) The concept of cross-flow filtration. In industrial use, membranes are incorporated ~n modules which are then configured ~n series and In parallel m various ways to produce a plant of the required separation capablhty 4. (b) The configuration for a small- scale batch process such as might be used ~n the laboratory or pilot plant work. (c) The module configuration ~n a three stage feed-and-bleed plant such as might be used on a full-scale b~otechnolog~cal plant. The operation ~s described m the mare text.

off continuously. Feed into the loop 1s controlled at a rate equal to the flow rates o f the permeate and concentrate. Large-scale plants use multiple stages, con- nected in series as ]llustrated in Fig. 2c. The advantages o f this type o f system include the continuous availabglty o f the retentate at the final concentratlOla required, and the need to sterihze the plant less frequently than batch processes.

The chemical engineering des]gn o f membrane processes requires a mathematical description o f the m e m - brane-permeation rate throughout the required process 4. There is, at present, no generally accepted set o f first principles that allows pre&ction o f membrane performance although, given sufficmnt data, there are general methods to describe the results. The aim of" this review is to focus on the present understanding of the main controlling parameters and to suggest how this knowledge should be used to optimize the operation o f existing and new processes.

Theories ofmicrofi ltration and ultrafiltration Numerous theoretical models for ultrafittratmon and

microfiltrauon have been proposed m the past 30 years, along with the ldentificauon o f new factors controlling flux or mass transfer through membrane.~. A few models, in which some significant and frequently

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Box 1. Typical operating

Ideally, cross-flow membrane processes would always operate at high, constant flux. Unfortunately, in practice, this is seldom the case. For both microfiltration and ultrafiltration, membrane flux decreases with time as illustrated in Fig. 1. The rate of decrease is less ff the cross- flow velocity ~s increased. Backflushmg, i.e. periodic forcing of permeate back through the membrane can, in some instances, enable good control of these decreases.

There have been many sophisticated treatments of cross-flow m~crofiltration. These include inertial migration models% shear-reduced hydrodynamic-convection models 6, shear-induced hydrodynamic-diffusion models', erosion models (scour and turbulent burst) 8 and friction-force models 9. All aim to describe how the build- ',p ol depcsl!s cn !he membrahe surface are controlled by :he hydrndynar..:i,,' (:,,;rK]ilions in the membrane module.

For those who are not specialists in the field, the basic operating patterns are best outlined in terms of the hydrodynamic resistance resulting from the build-up of deposited materials on the membrane surface. If all filtered materials remain on the membrane surface, then the flux will be given by:

c- O

g Q.

r -

..{3 E

a

b

~ c

Time

Figure 1 Schematic representation of the hme dependence of membrane permeatton rate (flux) during cross-flow membrane processmg. (a) Low cross-flow velocity, (b) increased cross-flow velocity, (c) with back-flushing.

g = (1/Am)(d F/dr) = IAPI/(P~+R¢)# = IAPI / [~+(e FCbA%)]# Eqn 1

where j is the flux, v the volume filtered, t the time, AP the applied pressure, ]~r-i the resistance of the membrane, R~ is the resistance of the deposited materials, (~ the specific resistance of the deposit, (:b the concentration of depositable materials ~n the process feed,, t m the membrane area and # the filtrate viscosity. For most b~ological materials, (x is a variable depending on the apphed pressure and time (the compressible deposit), so that the expression requires a iiunierical solution.

A useful method of allowing for the effects of cross-flow removal of depositing materials is to write:

.J = I._%Pl/tRr, + R~d-Rs,)lX Eqn 2

where -/¢,-d IS the resistance that would be caused by deposition of all filtered particles, and s{o~ is the resistance removed by cross-flow. It is then sometimes assumed that the removal of solute by cross-flow is constant, and equal to the con- vect~ve particle transport at sLeady state (=_/.~<S:i,) where.], ,(.h can be obtained experimentally, or from an appropriate model. In a number of cases, a steady state of filtration is never achieved. In such cases, it is possible to describe the time dependence of filtrabon by Introducing an efficiency factor [;, representing the fraction of filtered material remain- ing in the deposit rather than being swept along by the bulk flow. ThB gives:

R c = 13~l."(:b/sl,. , Eqn 3

Permeate

Membrane

Solute build-up

~'~ ~Cw F ~ - --I-- ~-

g - . -~ _Dd_ C_

8co 2____ ~5

o9

0

Distance from m e m b r a n e (Y)

Figure 2 Concentration polarization at a membrane surface.

where 0<13<l. Although deposition also occurs dur-

ing ultrafiltratlon, an equally important factor controlling flux is concentration polarization (Fig. 2). This results from the rejection of macrornolecules at the membrane front surface and their build- up until the rate at which they are transported back into the bulk liquid by molecular or eddy diffusion is matched by the rate at which they are brought to the membrane surface. Typical operating patterns for ultraflltrat~on are shown ~n Fig. 3a-c. The first model to account successfully for the patterns illustrated ~n Fig. 3b and c was the film model 12. The key equation of this model is.

J = !., In(('.'./(: b) Eqn 4


455

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patterns of membrane processes

a b C

" b

x

,T / Applied-pressure dtfference

••o•i Htgh ross-flow

ity

cross-flow ~ \ 7 velocity "" \ ~ \ J

In Gb Figure 3

Turbulent /

/ / ~ l o p e 0,8

Laminar /

~/~Slope 0,33

In (cross-flow velocity)

Typical dependence of membrane flux on (a) apphed pressure difference, (b) solute concentratton, and (c) cross41ow veloGy

where k. is a mass-transfer coefficient, c'~ is the solute concentration at the membrane surface and c b is the bulk-solute concentration. Thts equatton explatns the dependence of flux on Incb and, through the use of conventional correlations for the mass-transfer coefflctent (for example, Leveque for laminar flow and Dlttus-Boelter for turbulent flow), the dependence of flux on In(cross-flow velocity). However, this model can only explan the pressure mdependent region of F,g. 3:: tl,rc, ugh the fij.~.hr, r assumption that at high pressure a 'gel' of solute forms on the membrane surface .'. ih r .-( =-c::,n:q;m:. '.'.q(:re cg is the concentration of solute in the gel. For some proteins thts is a reasonable assumption, although for others it is rather dubious, as explained m the main text.

The most generally useful simple model of ultraflltration in the absence of depositton or adsorption is undoubtedly. the osmotic-pressure modeP 2. This begins with the expression:

j = (lAPl-lz~r/lyP, m~ Eqn 5

where A B is the osmotic pressure d~fference across the membrane. The osmotic pressure of concentrated solutions can be represented in terms of a polynomial:

lr[ = alC + a2 C2 + a3C3 Eqn 6

where al, a 2 and "3 are coefficients and c is the solute concentration expressed as a mass fraction, tn the present case, this is approximated as:

1~171 = H = aC~ Eqn 7

where (-. is the concentration at the membrane surface and n> l . The film-model expression (Eqn 4) Is used to cal- culate the value of ( ; . The use of such an expression ensures that the dependence of J on Incb and In(cross-flow vel- ocW) is correct. In addition, manipulation of the basic equation gives:

Pt//ol2,/'l = (R.~,ff + nlS II I /kH --(I,~.~,# + n(15nl-:lR,-,~)/k) 4 Eqn 8

which has asymptotes:

~,_iAfl-kr, I ~ (R,~,~) -t for I&PI-> 0 or I~111--; 0 and o]/ol..k/'l--~ 0 for I±PI - > ~ or I±PI >>_lR, ,~

hence correctly explaining the flux-pressure profiles of Fig. 3a.

These strnple rnodels have been constderably refined to allow for variation of wscosity and diffusion coefficient across the concentration polarized layer. Also the Lev~que and Dfitus-Boelter correlations (examples of Sherwood relations) often do not provide an exact correlation of the effects of cross-flow velocity. In a recent rewew v3, no fewer than 27 d,fferent Sherwood relations were given for turbulent flow of NewtonBn fluids tn pipes or ducts. Adding the relahons for non-Newtonian fluids as well as the relations for the lam,lar flow case. it becomes clear that choosing a relat~on- shtp that describes a certain system accurately ~s very difficult. Further, many of the re!at~ons used have been devel- oped not for membrane processes but for mass transfer ~n non-porous systems, or were derived from heat-transfer/mass-transfer analogies.

There ts also at present tntense tnterest ~n the use of complex flow patterns; for exampie, such as can be enduced by baffles and pulsed flow, or vortex flow. This can result m a constderable increase ~n the effective mass transfer co- efftc~ent and hence faster separation.

_ m

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occurring t:actors were identified and their significance quantified, have gained universal recognition. In other cases, models are adequate for some types of solute, but not for others. Often, it is inappropriate assump- tions of a particular model that lead to Failure in such cases. Some of the basic features of successful models arc described below; the relevant equations are presented in Box 1.

One of the mo~t important features ofultrafiltratlon, f}om a theoretical point ofvmw, is the concentration polanzation that results from the rejection of macromolecules at the su~acc of the membrane and thmr bmld-up until the rate at whmh they are transported back into the bulk lmqmd, by molecular- or eddy diffusion, ~s matched by the rate at which they are brought to the membrane sunCacc. It has been estab- lished that, under given cross-flow condiuons, there is often a tlm~ting flux for a particular solution, whmh is not exceeded when transmembranc pressure is increased. The flux at which this limiting concentration will be reached depends on the bulk-solute concentration, and on the mass-transfer coefficient taking solute back into the bulk flow, which depends on solution rhcology and hydrodynamics. During batch filtrauon, the solute concentration bmlds up and the flux dechnes.

As described m Box 1, this hrmting concentrauon was initmlly interpreted as a gclhng concentration (the concentration at which deposition of the solute occurs), controlled by the limiting solubihty of the solute. Th]s assumption is, in general, unrehable as it has been shown that the gelling concentration for a gwen solute can vary by as much as 50% with different membrane types. A more complete descrlpuon of the dependence of the membrane-permeauon rate on the applied pressure may be given by considering the effect of the osmotic pressure of the macromolecules. The osmotic-pressure model is the s~mplest model that can account for the mare process patterns of ultrafiltration. The most important of these are illustrated m Figs 1-3 in Box 1. The basic features o fp r c s su re~ux profiles (Box 1, Fig. 3) are:

(1) At low apphed pressure (]Ap]) , the slope of the pressure-flux prof le is similar to pure solvent flow. (2) As ]Ap[ increases, the slope dechnes and approaches zero at high pressure - essentially, the osmotic-pressure model explains the bruiting concentration as the concentration at whmch the osmotic- pressure difference across the membrane (Le. the difference between the osmouc pressure of the concentrauon-polarized solution at the membrane surface and the permeate) approaches the apphed- pressure difference across the membrane.

Through the mass-transfer correlations, the osmonc- pressure model explains the way m which the m e m - brane flux vanes with the cross-flow velocity (u); for example, at low cross-flow velocity (laminar flow), the flux (J) 0c u033, whereas at high cross-flow velocity, j oc u" s (although, in pracuce, many different depen- dencies have been tbund). This model, which has been

subjected to a number of refinements, is probably the

most generally usefial descnpuon of protein ultrafiltration.

The theoreucal descnpuon of the mlcrofiltrauon of protein soluuons is more complicated as, in many cases, colloids or particles such as cell fragments or intact ceils are also present. It is these larger-size materials, which are to be filtered out from the protein moleculcs that should be transported through the membrane. The retention of pamculate matenals results in the bmld-up of a 'filter cake' on the men> brane surface, even at high cross-flow velocities. The nature of such deposits is well-illustrated by the English use of the French term 'gateau', with its lmph- cauons of something sticky and imperrncablc. More technically, such deposits have a high resistance to fluid flow. In addition, some penetration of the membrane by particulate matenals wdl occur leading to par- ual pore blocking and loss of flux. Perhaps the worst type of pore blocking is that by particles of approxi- mately the same dimensions as the pore diameter. This may lead to complete blockage of the membrane pores and is descnbed as pore plugging.

Protein-membrane interactions The analysis outhned above gives a basic explanation

of the flux patterns 111 ultrafiltratlon and microfiltratlon. In pracuce, operating patterns frequently devi- ate f}om these patterns due to specifc interactions between solutes and the membranes. In contemporary ultrafiltration and ruicrofiltration processes, proteins are the solutes that have presented the most questions and parucular difficulties. In some studies, proteins have been found to be powerful adsorptive foulants, causing irreversible loss of membrane permeabihty 14'ts. There is also some suggemon that the specific bloac- tivity of protein molecules may be adversely affected by deconformation and denaturation dunng the filtration process. The inherent difficulties m quan- nfymg protein interaction with surfaces, the large number of variables influencing such interactions, and the astronomic number of possible configurations of a protein molecule in &fferent environments, make the quantitauve understanding of protein fouling and denaturauon an extremely difficult task.

Many experimental studies of protein ultrafiltrauon and m]crofiltrauon using stirred, unstirred and cross- flow systems have demonstrated that the filtrate fluxes obtained with protein soluuons are dramancally reduced compared with those obtained with the pure solvent under identical con&uons. Clearly, part of the flux decline is due to concentration polarizauon and protetn osmouc pressure, as described in Box 1. How- ever, an appreciable part of the decline may be duc to more-specific types of mteracuon between the pro- rein and the membrane surface, 1.e. adsorption and deposmon. Stu&es of such interacuons can be placed into one of two categories: (1) dnect measurements of the properties of proteins adsorbed or deposited during ultrafiltration and then characterized mdepeJ> dently; (2) redirect estimates of the properties of the


457

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Recipe Number 143: Blackforest filter gateau 'Is it time to add the cherries yet?'

protein layer, detcrmmed by fitting adjustable parameters In available models to experimental flux data. The majority of direct measurements have been made for ultrafittration. For example, the mass of bovine serum albumin (BSA) deposited on 100000 M W C O membranes during filtration has been determined by chemically desorbing the protein using sodium dodecyl sulphate 16. Protein coverages as high as 100 p, gcm -2 were determined: this is more than two orders of magnitude grcater than the rnonolayer coveragc typically obtained in the absence of filtration 17. The amount of protein deposited on thc suffacc was also a function of pH, with a maximum occurring at the isoelectric point of'the protein. Sire> lar results have been obtained with membranes that arc fully retentive to BSA (Rcf. 18), although with shghtly lower surface coverages.

Others have examined the structure of proteins deposited on both 10000 M W C O ultrafiltration membranes and 0.4b~m nncrofiltration membranes using scanmng electron microscopy (SEM). Results Indicated that BSA and [3-1actogtobulin formed shect- hke deposits on both the small- and large-pore membranes, whde y-globuhn formed granules that stacked into layers, creating a porous matrix. SEM studies have also been used to evaluate the thickness of protcin deposits during ultrafiltratlon, finding thicknesses of 0.5-1.0 Ixm for whey proteins, and a rangc of 5-30 Ixln for milk proteins.

Transmission electron microscopy (TEM) techniques have been used to examine protein-fouled ultrafiltration mcmbraneslg, >. hnmunochemIcal stare- mg has been applied to membranes to locate the pos- lt~on of protein foulants at the separation surface and within the mamx. It has been found that, as well as forming a thick fouhng layer on the membrane sur- t;~ce, protein foulants pass into the mamx. The power- t~l new techmque of atomic-force microscopy (AFM)

is providing detailed knowledge of the structure of both ultrafiltranon and microfiltration mcln- branes21 23. It has the tremendous advantage of allow- mg high-resolution investigauon m air and solvents and, thus, is very well-stated to the study of m e m - brane-protein interactions. Work in progress may well lead to radical reformulation of the effects of such mtcractions on flux patterns.

The formation of protein deposits of low pcr- meability on ultrafiltration membranes has also been deduced fi'om indirect measurements24; for example, from saline-flow measurements. It has been found that the hydraulic permeability at}er protein ultrafiltration decreased with increasing transmembrane pressure drop, suggesting that the protein behaved as a corn- pressible porous medium. The hydrauhc permeability after protein ultrafiltration has also been found to be a function or" the solvent properties, most notably increasing with decreasing ionic strength 17. In addition, the pcrmeabmhty of a deposited protein layer decreases with tImc. The thickness of protcin layers deposited on membranes can be evaluated indirectly from filtranon theory and a mass-balance. Estimates of the deposited thickness of BSA layers range from 1-5 urn For short filtration times, to as much as 150/~m for long times 24,2~.

From a technological point of view, the interaction of proteins with mlcrofiltration membranes is a very significant phenomenon. However , it has been less well-stu&ed than the interaction of" proteins with ultrafiltratIon membranes, where protein molecules are normally retained at the front surface of the membrane. Most stu&es have measured adsorption isotherms or adsorption kinetics, usually by simple &f- fusion fiom solunon. A number of factors have been shown to be unportant in determining the amount of protein adsorbed, including the mcmbrane surface- chemistry, the protein structure, the magnitude and

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polarity of both protein- and surface charge, and the degree of hydration of the protein. Manufacturers' literature also contains data relating to the adsorption of proteins by membranes. However , experimental protocols are not normally reported, making it difficult to assess the data. Nevertheless, the characteriz- ation of a membrane as ' low protein-binding' is seen as an important advantage, especially if the protein is a small volume, high-value speciality product, or IS in very dilute solution where predicted losses due to adsorption may have an Important influence on the overall yield of the product.

However, ~t is being increasingly realized that deter- ruination of protein binding at zero apphed-shcar conditions, where diffusion is the sole transport process, gives an incomplete assessment of" the effects of pro- tern-membrane interactions during processing. For example, measurement of the convective flow of pure BSA solutions through microfiltratlon membranes shows a continuous decrease m flux with time under constant applied pressure 2~', In some cases, this can be explained in terms of the deposition of"protein on the front face of the membrane. However, a continuous decrease also occurs when there is no deposition or concentration polarization on the front face of the membrane. Such decreases are much greater than can be explained by protein adsorption, which is generally hmitcd to monolayer coverage. Furthermore, they are independent o f the protein-binding nature of the membrane, being almost Identical for membranes with either low or high protein-adsorption characteristics under static conditions. Such decreases have been interpreted in terms of" protein deposition resulting from shear-induced distortion of molecules on passage through mlcropores. The decrease in flux with time can be descnbed quantitatively as a decrease in pore volume due to deposition of protein on the walls of the pores. Under solution conditions where enzymes are slightly unstable, a small but permanent loss of enzyme actwlty on passage through mlcrofiltratlon membranes can be measured 27. Some permanent changes in secondary structure have also been shown by circular dichroism and Fourler-transf"ormed mfrared spectroscopy 2s. However, such changes are easily minimized by careful control of" pH.

Some current developments Although microfiltrauon and ultrafiltratIon have

been the subjects o f wide-ranging investigations, there are still a number of important phenomena controlhng performance that have been rather neglected. Two key examples are colloidal intcracuons, both solute-solute and solute-membrane, and hydrodynamic effects m membrane pores.

Solute-solute interactions result from the obser- vauon that all materials dispersed in aqueous mcdia acqmre a surface charge. Such surface charge is an important determinant of the solution properties of proteins, protein aggregates and m~crobial cells. As a result, the surf:ace-charge properties of both m e m - branes and dispersed materials are Important consider-

ations m mlcrofiltranon and ultrafiltratlon, even though these processes arc often considered only m terms of the size of materials being separated.

A simple case is that of the adsorption of the protein BSA at an inorganic membrane. The pI of BSA is at - pH4 . 9 , so that it has a net negative charge above tins pH, and a net posinve charge below It. The membrane charge affects the membrane surface potential and hence the zeta-potential (the potential at the plane of shear). Figure 3a shows data for the surface electrical properties of a capillary-pore inorganic microfiltrat~on membrane (Anopore, ().2Dm) 29. These properues have been calculated fFom measurements of the rate of electro-osmosis across the membrane under the influence of the apphed electric ficld. These membranes have a high negative zeta-potential at high pH, an isoelectrlc point at pH4.5 and a moderately pos> rive zeta-potenual at low pH. Figurc 3b shows the amount of protein adsorbed on Anopore 0.21*m membranes as a function of pH (P,,ef. 30). The data show the interplay between electrostatic- and hydration-controlled interactions. At pH6.5, both membrane and protein bear a net negative charge. The charge on the protein decreases as the protein approaches its lsoelectrlc point, and the amount of adsorbed protein increases. This is because the degree of hydration is minimum at the isoclcctric point, allowing short-range electrostatic forccs (van der Waals) or hydrophoblc mteracuons to COlhe into play. Between pH4.9 and 4.5, the protein has a net posi- tive charge and the membrane a net ncgatwe charge. However, there is no increase in protein adsorption, but rather a small dccrease, which reflccts an increase In effective protein size due to Increasing hydrauon. At pH values below 4.5, both protein and membrane are posmvely charged. Here, the amount of protein adsorbed decreases rapidly, showing that electrostanc repulsion is dominant.

It has been menuoned previously that the hydrauhc resistance of protein deposits depends on the lomc strength of the permeating fluid. Such effects may bc explained quantitatively, in terms ofelectrostauc mter- acnons due to the repulswe interactions of the double layers around the proteins and to attractwe van der Waals forces. Hence, the resistance of such deposits dcpends not Olfly on the size and packing of thc materials deposited, but also on their mteraction energy, as such interactions control thc spacing between material in the deposited layer. Thus, calculations of lnterac- non energies allow prediction of the hydrauhc resistance. Such calculations show that the low-pressure filtration of particles of the size of protein aggregatcs with high zeta-potential may result m deposit resist- ances of less than 5% of those expected, if the pamcles m such deposits were in close contact 31. Indeed, van- ation of zeta-potential and apphed pressure gives a variation in resistance of a factor of" 1 6, even at constant particle size and ionic strength. This shows clearly the importance of operating condluons (pH, lomc strength, applied pressure) in determining the resistance of deposits formed on membranes.

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A second, neglected p h e n o m e n o n controlhng membrane performance ]s the convectwe-dlf fus ive transport o f solutes or particles that have entered pores. This is quite distinct f rom adsorpnon or deposition o f parncles on pore walls. Many studies have been car- tied out on the &ffusion of solutes through pores with no applied pressure. In such cases, it is observed that the apparent diffusion coefficient o f a solute in a porc becomes much lower than that in bulk solution if the pore dimensions are o f the same order as those o f the solute. Such solute transport is described as hindered or restricted 32. It is much less generally recognized that the pressure-driven convecuve-di~/iasive transport o f solutes through pores is also hindered and that the presence o f solutes in pores that are neither adsorbed nor deposited, but continue to move with the bulk flow, gives rise to an extra pressure loss, or else a lower flux at constant pressure. Such hindered transport in pores is an important reason why the rate ofmlcrof i l - trauon o f protein solutions can never be carried out at a rate equal to the water-permeation rate o f the m e m - brane, under otherwise comparable conditions.

Considerat ions for o p t i m i z i n g m e m b r a n e per formance

Membrane microfiltratlon is one o f the most effec- nve ways o f separating proteins from materials o f larger size, such as microbial cells and celt debris. Membrane ultrafiltration is the most effective means o f concentrating protein solutions. In both cascs, there is a reasonable understanding at the macroscopic level o f the dependence o f the flux patterns on the major operating parameters o f apphed pressure and hydrodynamics.

A major goal for membrane technologists is to develop predicuve descriptions o f flux patterns, based on a rigorous understanding o f the underlying physico-chemical phenomena. This is the subject o f substantial research on an international basis. The emphasis is on quantitative analysis o f the micro- hydrodynamics and mtcrfacial aspects o f membrane processes. These current developments are likcly to lead to fully quantitative and predictive descripnons o f the operating characteristics o f membrane processes.

From existing knowledge, it is possible to define a number o f guidelines for the successful processing o f proteins by membranes:

• Select the membrane carefully. Much emphasis 1s rightly placed on the composiuon o f the polymer or inorganic oxide used. This is justified due to the importance o f interracial mteracuons In membrane processing. However , pore size or M W C O and pore morphology are equally important in maintaining high fluxes. It is crucially important to avoid pore plugging, and rapid changes in pore dimensions create points o f high shear that may damage proteins and facilitate their deposmon inside pores. • Cont ro l the solution con&tions carefully. Opt i - mization o f pH and lomc strength can minimize adsorption, deposinon and damage to proteins. Such

4 "

4o -80

-120 20 30 4'0 5'0 610 710 810

pH

4.0

E 3.0- g

~ 2 0 -

C

• ~ 10 - 13- /

3.0 0.0

, • 6~0 2.0 40 50 0 pH

91o

Figure :3 (a) The dependence of zeta-potential on pH for a capillary pore inorganic membrane (Anopore, 0.2 i~m). The c~rcles represent a calcu latlon based on Smoluchowskl's classmal expression and the squares depict a numerical calculation. (b) The dependence of the amount of BSA adsorbed on pH for a capillary pore inorganic membrane (Anopore, 0.2 i~m).

optmuzat ion can also gwe order o f magnitude decreases in the hydraulic resistance o f deposits on the front face o f the membrane. These are the most frequently neglected aspects o f effective membrane use. • Select the hydrodynamic conditions with care. It is important to create effectwe mass transport at the membrane surf'ace. N e w module designs with effective baffling and variable-flow regmles can make very efiCectlve use o f energy Input. • Operate at low applicd pressure. 1)cpos]t]on m pores and the hydraulic resistance o f deposits on the front face o f the membrane increase substantially at higher pressures. Although high pressure gives initially higher filtrauon rates, lower-pressure operation often results in higher overall rates o f filtration as the effects o f such membrane fouling are minimized. Particular attention should be given to the start-up o f processes. A gradual increase m operating pressure mimmizes flux losses.

These guidehnes may sccm simple, but they arc not simply rules resulting from operating experience T h e y have a firm basis in the science underlying m e m - brane processes.

TIBTECH NOVEMBER 1993 (VO[

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reviews

References 1 Rautenbaclh R and Albrecht, R (1989) Membrane Protessr~, Wlle'v 2 Kennedy, J F and Cabral,J M S (eds)(1993)Recol,ery Proce~sesjbt

BMoglcal Materials, Wiley 3 Mulder, M (1991) Basic Pnnclples ofMembr, me Tedmology, Kluwer

Academic 4 Bowem W R (1991) m Chemical Engineermj.~, 4th edn, Vol 2

(Coulson, J M and P.lchardson,J F, eds), pp 858-891, Pergamon 5 Altena, F W and Belfort. G (1984) Chem Eng &i 39, 343-355 6 Daws, R H and Blrdsell, S A (1987) Chem En,,~ Commml

49, 217-234 7 P.omcro, C A andDavis, R H (1988).J Memlmme&I 39, 157 185 8 Fane. A G,Fell , C J D and Nor. M T (1982)hid Chem Eng

yubdee Syrup 73, C1-12 9 Blake, N J , Cummlng, 1 W and 8treat, M (1900) m P~oceedlno~s of

the Vth !4"odd Filhatton Col12~e.% Nice, France, pp 579 585, Soci4t4 Prangalse de Filtration

10 Fane, A G (1986) m Pw2res~ .t Filhatwn and Separatton, Vol 4 (Wakeman, R J , ed.), pp 101 179, Elsevmr

11 Mlchaels, A S (1968)Chem hug Pw2 64, 31 43 12 Wijmans, J G., Nakao, S and Smoulders, C A (1984)J Membrane

S~1 20. 115-124 13 Gekas. V and Hallstr6m. B (1987)J Membrane &i. 30, 153-171) 14 Mmrcles, M. Annar, P and Sanchez, V (1991)J, Memlmme Sa

56, 13-28 15 Hanemaauer, J. H , Robbertsen, T , van den Boomgaard, T H and

Gunmck, J W (1989)o/ Membrane Sol. 40, 199 217 16 Fane, A G (1983)~ Sep Process Ted,. 4, 15 23 17 Matthmsson, E (1983)3" Membrane &~ 16, 23-36

18 Sukl, A, Fanc, A (; and Fell, C J D (1984) J Membrane S. 21,269 283

19 Sheldon,J M (1991)j Membrane Sd. 62, 75 86 20 Sheldon, J M., Reed, 1 M and Hawas, C 14. (1991)J Membtane

Sa 62, 87 102 21 Dietz, P , Herrmanm K-H, lnackei, O , Lehmann, H-D and

Hansma, P K (IC)92)Soc Photo-optical lnmum En2 Pwc. Set. 1639, 186-197

22 Chahboun, A. Coratoger, R , Ajustron, F and Beauvlllam,J (1992) UltmmioosColly 4I, 235-244

23 Bessl&res, A , Meireles, M , Almar, P , Sancbcz, V, Coratger, R and Beauvillam, J (1992) R&ents P~og*A en Genie des Pwc&l&s 22, 111 116

24 Chudacek, M W and Fane, A G (1984) J Membrane Su 21,145-160

25 1Kelhanian, H , Robcrtson, C R and Mlchaels, A S (1983)J Mem- brane Sci 16, 237 258

26 Bowen, W R. and Gan, Q. (lq91) BIotedmol Bloen.q 38, 688-696 27 Bowem W R. and Gan, Q. (1992) Btotedmol Bioeng 40, 491-497 28 Franken, A C M.,SltIys, J T M , C h e u , V,Fane, A G and Fell,

C J D (1990) m Proceedtngs of the I/?h Yi'orld Fdtratlon Con2ress, Nice, France. Vol 1, pp 207~13, SocI&~" Franqalse de Flltratmn

29 Bowen, W P. and Hughes, D T (1991)_/ Colloid lnte~ace Sol 143, 252 265

30 Bowen, W IK and Hughes, D T (1991)J Membrane So 51, 189 200

31 Bowen, W R (1993) In Membrane~, Proees~e~ aim Apphcatlons (Hemandez-Gmlenez, A, ed ), pp 155-174, Umversldad de Valladohd

32 Deen, W M (1987)Am ln~l Che.i Eng d 33, 1409 1425

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Understanding flux patterns in membrane processing of protein solutions and suspensions

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