diafiltration for desalting or buffer exchange

MAY 2003 BioProcess International 43

DD iafiltration is anultrafiltrationmembrane techniquefor completelyremoving, replacing, orlowering the concentration of saltsor solvents from solutionscontaining proteins, peptides,nucleic acids, and otherbiomolecules. The processselectively uses permeable (porous)membrane filters to separate thecomponents of solutions andsuspensions based on theirmolecular size. Smaller moleculessuch as salts, solvents, and waterpass freely through theultrafiltration membrane, whichretains the larger molecules.

CONCENTRATIONThe solution retained by amembrane is known as concentrateor retentate. The solution thatpasses through a membrane isknown as the filtrate or permeate. Amembrane for concentration isselected based on its rejection

characteristics for the mixture that isto be concentrated. As a generalrule, the molecular weight cut-off(MWCO) of a membrane should bea third to a sixth the molecularweight of the molecule to beretained. This is known as the 36Rule, which ensures completeretention. The closer the MWCO isto that of the sample, the greaterthe risk for some product lossoccurring during concentration.That risk increases if diafiltrationwill also be used because the relativeloss depends on the total volume offiltrate generated.

Membrane flux rate (defined as thefiltrate flow rate per unit area ofmembrane) is related to pore size.The smaller the pores, the lower theflux rate for the same applied

pressure. Therefore, when selectinga membrane for concentration ordiafiltration, consider the time factorversus product recovery. In mostbiological applications, recoveryoutweighs the time consideration.Increasing the amount of membranearea used can also reduce theprocess time.

Figure 1 provides an example ofconcentration. The sample is placedin a device containing a suitableultrafiltration membrane that willretain the large molecules. Pressureis applied until half the volume haspassed through that membrane.Large molecules are retained in halfthe original volume (concentrate),which also contains half of the saltmolecules. The filtrate contains theother half of the salt molecules but

Diafiltration for Desalting or Buffer ExchangeLarry Schwartz

PRODUCT FOCUS: ALL BIOLOGICALS

PROCESS FOCUS: DOWNSTREAMPROCESSING

WHO SHOULD READ: PROCESSDEVELOPMENT, MANUFACTURING

KEYWORDS: ULTRAFILTRATION,DIAFILTRATION, BUFFER EXCHANGE,DESALTING, PROTEIN CONCENTRATION

LEVEL: INTERMEDIATE

A typical development laboratory system comprising a Centramate holder with aperistaltic pump and pressure gauges, valves, and tubing. PALL CORPORATION(WWW.PALL.COM)

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44 BioProcess International MAY 2003

none of the large molecules.Therefore, the large molecules areconcentrated as liquid and salt areremoved. The salt-molecule-to-volume ratio in the concentrateremains constant, so the ionicstrength of the concentratedsolution remains relatively constant.

Diafiltration, the process ofwashing the remaining salt outwith water, can subsequently reducethe ionic strength of a concentrate(retentate) solution. This isessentially a dilution processperformed in conjunction withconcentration. Water is added whilefiltrate is removed. If the washingsolution is another buffer instead ofwater, the new buffer salt willreplace the initial salt in the sample.For simplicity, the examples hereinuse a direct-flow filtration devicesuch as a centrifugal concentrator.But the same principles apply tocross-flow filtration devices such ascassettes and hollow fibers.

BENEFITS OF DIAFILTRATIONOther techniques used for saltremoval or buffer exchange (such asmembrane dialysis and column-based gel filtration) can be effectivebut have certain limitations. Dialysisprocedures can take several days,require large volumes of water forequilibration, and risk product lossthrough manual manipulation ofdialysis bags. Gel filtration dilutesthe sample and often requires anadditional ultrafiltration step toconcentrate it back. Adding steps to

a process can lead to the possibilityof sample loss or contamination.

With diafiltration, salt or solventremoval and buffer exchange can beperformed quickly and conveniently.Another advantage of usingdiafiltration is concentration ofsamples on one system, minimizingthe risk of sample loss orcontamination. Diafiltration isperformed in three main ways: bycontinuous diafiltration and bydiscontinuous diafiltration throughsequential dolution or volumereduction. Although the end resultmay be the same overall, the time

and volume required to completethe process varies considerably. It isimportant to understand thedifferent methods used and when tochoose one over the other.

CONTINUOUS DIAFILTRATIONThe technique of continuousdiafiltration (also referred to asconstant volume diafiltration)involves washing out the originalbuffer salts (or other lowmolecular-weight species) in the retentate(sample) by adding water or a newbuffer to it at the same rate filtrateis generated. As a result, the

TTaabbllee 11:: Continuous (constant volume) diafiltration

Removal of Small Molecules

Diafiltration Permeability 100% Permeability 75%Volumes Rejection Coefficient 0 Rejection Coefficient 0.25

1 63% 53%2 86% 77%3 95% 89%4 98.2% 95%5 99.3% 97.6%6 99.7% 98.9%7 99.9% 99.4%8 99.7%9 99.9%

Note: 0% rejection salts, solvents, buffers, etc.; 25% rejection molecules lower in molecular weight thanthe molecular weight cutoff of the membrane, but bigger than salts

FFiigguurree 11:: 2 concentration of a sample mixture by ultrafiltration. Large circles representmolecules that are bigger than the pores in the membrane, and small circles representmolecules (such as salts or solvent) that are smaller than those pores.

AnotherAADDVVAANNTTAAGGEEof usingdiafiltration isconcentration ofsamples on onesystem, minimizingthe risk of sampleloss orcontamination.

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retentate volume and productconcentration do not change duringthe diafiltration process. If water isused for diafiltering, salts will bewashed out and the retentateconductivity lowered.

If a buffer is used for diafiltering,the new buffers salt concentrationwill increase at a rate inverselyproportional to that of the molecularspecies being removed. The amountof salt removed is related to thefiltrate volume generated relative tothe retentate volume. The filtratevolume generated is usually referredto in terms of diafiltration volumes(DVs).

A single DV is the volume ofretentate when diafiltration begins.

Liquid is added at the same rate asfiltrate is generated, and when thevolume of filtrate collected equalsthe starting retentate volume, 1 DVhas been processed. Usingcontinuous diafiltration, over 99.5%of a 100% permeable solute can beremoved by washing through sixvolumes (6 DV) with the buffer ofchoice. Molecules that are largerthan salts and solvents but smallerthan the pores in the membrane alsocan be washed out. Thepermeability of such molecules,however, may be less than 100%. Ittakes more liquid (more DVs) tocompletely wash a partiallypermeable molecule through themembrane than it does to remove a100% permeable molecule from amixture. Typically, the larger themolecule, the lower its permeabilityand the greater the wash volumerequired. The permeability of aparticular molecule through aspecific membrane can bedetermined by measuring theconcentration of that molecule inthe filtrate compared to itsconcentration in the retentate underspecified conditions (Equation 1).

Permeability is often described interms of the rejection coefficient ofthe membrane (its ability to holdback or reject a given molecule frompassing through). Thus, seeEquation 2.

A rejection coefficient of 1 equals0% permeability. A rejectioncoefficient of 0 equals 100%permeability. Permeability is affectedby such factors as transmembranepressure (TMP), crossflow rate,retentate concentration, pH, ionicstrength, and gel layer formation(concentration polarization).Therefore, the permeability maychange over the course of thediafiltration process.

Table 1 shows the relationshipbetween permeability through amembrane and the number ofdiafiltration volumes required forremoval of permeating species. Asnoted above, greater volumes ofbuffer are required to removemolecules that are partially retained.To remove 99.9% of a molecule thatis 25% permeable to the membranerequires 9 DVs, whereas for a 100%permeable species, only 7 DVs arerequired to achieve the same

A Minimate TFF Capsule device fordevelopment and processingapplications using small samplevolumes. Samples can be concentrateddown to about 5 mL and diafiltered.PALL CORPORATION (WWW.PALL.COM)

TTaabbllee 22:: Salt reduction from sample using volume reduction or constant volume diafiltration

2 Volume Reduction Constant Volume

Diafiltration 100% Permeability 75% 100% Permeability 75%Volumes 0% Retention 25% 0% Retention 25%

1 50% 41% 63% 53%2 75% 65% 86% 77%3 88% 79% 95% 89%4 94% 88% 98.2% 95%5 96.9% 93% 99.3% 97.6%6 98.4% 95.6% 99.7% 98.7%7 99.2% 97.4% 99.9% 99.4%8 99.6% 98.4% 99.7%9 99.9% 99.4% 99.9%10 99.9% 99.4%

Note: 0% rejection salts, solvents, buffers, etc.; 25% rejection molecules lower in molecular weight thanthe molecular weight cutoff of the membrane, but bigger than salts

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% Permeability (ConcentrationFILTRATE ConcentrationRETENTATE ) 100

EEqquuaattiioonn 22

Rejection Coefficient 1 (ConcentrationFILTRATE ConcentrationRETENTATE)

Typically, the largerthe MMOOLLEECCUULLEE,,the lower itspermeability andthe greater thewash volumerequired.

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removal.

DISCONTINUOUS DIAFILTRATIONSequential Dilution. Discontinuousdiafiltration by sequential dilutioninvolves first diluting a sample withwater or a replacement buffer to apredetermined volume. The dilutedsample is then concentrated back toits original volume by ultrafiltration.The process is repeated until theunwanted salts, solvents, or smallermolecules are removed. Eachsubsequent dilution andconcentration step removes more

small molecules. As shown in Figure 2, the sample is generallydiluted with an equal volume ofbuffer (1 DV). Adding volumes 1DV will be less effective. Forexample, diluting the sample with 5 DVs and concentrating back willreduce the salt by only 80%compared with 88% for 2 volumereduction or sequential dilution.Diluting the sample usually lowersviscosity, which may increase thefiltrate flux rate.

Volume Reduction. Discontinuousdiafiltration by volume reduction reverses the sequential dilution

DIAFILTRATION DEFINITIONS

Diafiltration: Diafiltration is atechnique that uses ultrafiltrationmembranes to completely removeor to lower the concentration ofsalt or solvent or to replacebuffer salts from solutionscontaining proteins and otherlarge molecules.

Diafiltration Volume: Onediafiltration volume equals theinitial volume in which themolecule of interest is suspended.The number of diafiltrationvolumes required depends onwhether the permeating molecularspecies is freely passing (salts,buffers, solvents) or partiallyretained.

Continuous Diafiltration: Thetechnique of continuousdiafiltration (also referred to asconstant volume diafiltration)involves washing out the originalbuffer salts (or otherlowmolecular-weight species) inthe retentate (sample) by addingwater or a new buffer to theretentate at the same rate asfiltrate is being generated.

Discontinuous Diafiltration bySequential Dilution involves firstdiluting the sample to apredetermined volume, and thenconcentrating that sample back toits original volume with water orreplacement buffer. This isrepeated until the unwanted salts,solvents, or smaller molecules areremoved. Each subsequentdilution removes more of them.

Discontinuous Diafiltration byVolume Reduction involves firstconcentrating the sample to apredetermined volume, thendiluting that sample back to itsoriginal volume with water orreplacement buffer. This isrepeated until the unwanted salts,solvents, or smaller molecules areremoved. Each subsequentconcentration and dilutionremoves more of them.

FFiigguurree 22:: Discontinuous diafiltration (sequential dilution). Large circles represent moleculesthat are bigger than the pores in the membrane, and small circles represent molecules (suchas salts or solvent) that are smaller than those pores.

FFiigguurree 33:: Discontinuous diafiltration (sequential dilution). Large circles represent moleculesthat are bigger than the pores in the membrane, and small circles represent molecules (suchas salts or solvent) that are smaller than those pores.


procedure. A mixture is firstconcentrated to a predeterminedvolume and then diluted back to itsoriginal volume with water or areplacement buffer. This is repeateduntil the unwanted salts, solvents, orsmaller molecules are removed.Each subsequent concentration anddilution removes more smallmolecules (Figure 3).

After the last buffer addition, thesample is generally concentratedbefore analysis or before the nextpurification step is performed. Afterdiafiltration by either method(discontinuous 2 volumereduction or sequential dilution),the final product is at the samevolume and concentration as whendiafiltration started. The saltconcentration has been equallyreduced in both examples.However, the volume of diafiltrationbuffer used by the volume reductionmethod reduced by the initialconcentration step was half thatused in sequential dilution. A DV is

equal to the volume where dilutionoccurs. Therefore, half the volumewas required.

That being the case, it wouldseem that concentrating beforediafiltration by eitherdiscontinuous sequential dilution orconstant volume diafiltration should reduce the requireddiafiltration buffer volume and savetime. And in most cases this is true.The factor we have not accountedfor is filtrate flux rate, which equatesto process time. As the productbecomes concentrated, viscosityincreases, and the filtrate flux ratedecreases. The filtrate flux rate variesinversely as the log of theconcentration factor:

J k ln (CGCB)

where J filtrate flux rate, k constant, ln natural log, CG gel layer concentration, andCB retentate (bulk flow)concentration. This becomes verysignificant as the CB increases abovea few percent and is dependent onthe characteristics of the specificmolecules making up the samplemixture. Although it might takesignificantly less volume to diafiltera concentrated sample, it could takeconsiderably more time comparedto a less concentrated sample.Simple protocols are available to

find optimum conditions thatmaximize productivity.

WHICH TECHNIQUESHOULD BE USED?When deciding which technique touse and where in the downstreamprocess to perform diafiltration,consider the following factors:

Initial sample volume,concentration, and viscosity

Required final sampleconcentration

Stability of the molecule ofinterest at various concentrations

Volume of buffer required fordiafiltration

Total processing timeReservoir size availableEconomics.The choice of which method to

use must be based on severalcriteria. Scale is an importantconsideration. What we do atlaboratory scale may be verydifferent than at process scale,especially if the process isautomated. At lab scale,discontinuous diafiltration is oftenused for its simplicity. Continuousdiafiltration requires a pump orspecial equipment to add thediafiltration solution at a constantrate. Both techniques can beautomated for process applications.

If we eliminate the equipmentissue and focus on the process itself,we can compare the differences.Ionic strength, buffer composition,and stabilizer concentration canaffect stability of the sample.Diafiltration may remove salts orstabilizing molecules, resulting inprotein product denaturation andaggregation. The process ofconcentrating and diluting a proteinsolution can affect molecularinteractions to cause denaturation oraggregation, subsequentprecipitation, and product loss. It isnecessary to evaluate the effect of

FFiigguurree 44:: Determination of the CG value for a product.

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Process Time Filtrate Flow Rate Volume

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ln (CG CR) 1 or CROPTIMUM CG e 0.37 CG

Although it mighttake less volume todiafilter aconcentratedsample, it couldtake more timecompared to a lessconcentratedsample.


concentration on the product itselfto determine where diafiltration isbest performed relative toconcentration effects. Continuousdiafiltration offers an advantage overdiscontinuous diafiltration in thatthe retentate concentration remainsconstant. It is often seen as a moregentle process.

BEFORE OR AFTER CONCENTRATION?We have already seen thatconcentrating a sample first cansignificantly reduce the volume ofdiafiltration solution required. Wehave also seen that continuousdiafiltration takes less volume thandiscontinuous diafiltration withsequential dilution. Therefore, if thesample is first concentrated to itsrequired final concentration andthen continuous diafiltrationperformed, acceptable results shouldbe obtained.

However, above a certainconcentration, filtrate flux rates maybecome prohibitively slow. It mayactually take longer to diafilter aconcentrated sample than it would ifthe sample were first diluted toreduce its concentration. In thissituation, even though continuousdiafiltration of the diluted samplerequires a greater diafiltrationvolume, the total processing timewould be lower because of the fasterfiltrate flux rate (Equation 3).

In general, the optimumretentate concentration for

performing (continuous)diafiltration can be determined as inEquation 4, where CG gel layerconcentration, CR retentateconcentration, and CROPTIMUM highest retentate concentrationwhere diafiltration should beperformed (1).

The CG value for a samplemixture can be determined fromexperimentation by concentrating asample on a membrane whilerecording and plotting data forfiltrate flux rate compared with logconcentration (concentrationfactor). Then a curve can beextrapolated to filtrate flux rate 0.The CG value will be the same forthis particular product regardless ofthe starting concentration or filtrateflux rate.

In Figure 4s example, the CGvalue is a concentration factor ofapproximately 33. Therefore, theoptimal concentration factor toperform diafiltration would be 0.37 CG 12.2. If the startingproduct concentration is 5 mg/mL,then diafiltration should beperformed when that concentrationreaches 61 mg/mL. If the finalconcentration will be less than 61mg/mL, then diafiltration shouldbe performed after concentrationunless it is necessary to remove aspecific molecule first.

The ultrafiltration productselected may dictate your choice ofcontinuous or discontinuousdiafiltration. Stirred cells andcentrifugal devices are best suitedfor discontinuous diafiltrationbecause of their mode of operation.Tangential flow devices have theadvantage of being useful for eitherdiafiltration technique.

A USEFUL STEPIN DOWNSTREAM PROCESSINGDiafiltration is a fast and effectivetechnique for desalting or bufferexchange of solutions. It can beperformed in a continuous ordiscontinuous mode. Continuousdiafiltration usually takes lessvolume to achieve the same degreeof salt reduction as discontinuousdiafiltration with sequential dilutionand can be easier to perform.

Continuous diafiltration is alsoperceived as a kinder and gentlerprocess on active biomolecules.

On the other hand,discontinuous diafiltration withvolume reduction requires lessvolume than continuousdiafiltration. Concentrating a samplemixture before diafiltration usuallyreduces the required filtrate volumeand saves time. However, if sampleviscosity becomes too great, thefiltrate flux rate will decrease, whichcan increase process timessubstantially. Determining the CGfor your sample can help answer thequestion: At what concentrationshould I perform diafiltration?

Highly selective polyethersulfone(PES) microfiltration andultrafiltration membranes withnarrow pore size distribution andhigh flow rates provide fastprocessing time and efficientseparation for diafiltration. Initialvolumes from a few milliliters up tothousands of liters can be processedusing tangential flow filtration. TFFsystems allow easy addition ofmembrane surface area, providingthe flexibility to reduce processingtimes or scale up your process. Therange of devices and systemsavailable provides scalability as wellas the flexibility to work with almostany sample volume.

REFERENCE1 Beaton, NV; Klinkowski, PR.

Industrial Ultrafiltration Design andApplication of Diafiltration Processes. J. Separ. Proc. Technol. 1983, 4(2) 110.

Larry Schwartz is Senior TechnicalManager at Pall Life Sciences, 516-801-9076, fax 516-801-9548;[email protected],www.pall.com.

Stirred cells andcentrifugal devicesare best suited fordiscontinuousdiafiltration;tangential flowdevices have theadvantage of beinguseful for eitherdiafiltrationtechnique.

diafiltration for desalting or buffer exchange

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