Diafiltration for Desalting or Buffer Exchange

1 MAY2003 BioProcess International43 DDiafiltration 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.

2 The solution thatpasses through a membrane isknown as the filtrate or permeate. Amembrane for concentration isselected based on its rejectioncharacteristics 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 3 6 Rule, which ensures completeretention. The closer the MWCO isto that of the sample, the greaterthe risk for some product lossoccurring during risk increases if diafiltrationwill also be used because the relativeloss depends on the total volume offiltrate generated.

3 Membrane flux rate (defined as thefiltrate flow rate per unit area ofmembrane) is related to pore smaller the pores, the lower theflux rate for the same appliedpressure. Therefore, when selectinga membrane for concentration ordiafiltration, consider the time factorversus product recovery. In mostbiological applications, recoveryoutweighs the time 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 molecules are retained in halfthe original volume (concentrate),which also contains half of the saltmolecules.

4 The filtrate contains theother half of the salt molecules butDiafiltration for Desalting or Buffer ExchangeLarry SchwartzPRODUCTFOCUS:ALL BIOLOGICALSPROCESSFOCUS:DOWNSTREAMPROCES SINGWHOSHOULDREAD:PROCESSDEVELOPMENT,MAN UFACTURINGKEYWORDS:ULTRAFILTRATION,DIAFI LTRATION, Buffer Exchange , Desalting ,PROTEIN CONCENTRATIONLEVEL:INTERMEDIATEA typical development laboratory system comprising a Centramate holder with aperistaltic pump and pressure gauges, valves, and tubing. PALLCORPORATION( )44 BioProcess InternationalMAY2003none of the large , 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.

5 Diafiltration , the process of washing 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 simplicity, the examples hereinuse a direct-flow filtration devicesuch as a centrifugal the same principles apply tocross-flow filtration devices such ascassettes and hollow OFDIAFILTRATIONO ther techniques used for saltremoval or Buffer Exchange (such asmembrane dialysis and column-based gel filtration) can be effectivebut have certain limitations.

6 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 toa process can lead to the possibilityof sample loss or contamination. With Diafiltration , salt or solventremoval and Buffer Exchange can beperformed quickly and 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.

7 Although the end resultmay be the same overall, the timeand volume required to completethe process varies considerably. It isimportant to understand thedifferent methods used and when tochoose one over the technique of continuousdiafiltration (also referred to asconstant volume Diafiltration )involves washing out the originalbuffer salts (or other low molecular-weight species) in the retentate(sample) by adding water or a newbuffer to it at the same rate filtrateis generated. As a result, theTTaabbllee 11::Continuous (constant volume) diafiltrationRemoval of Small MoleculesDiafiltrationPermeability 100%Permeability 75%VolumesRejection Coefficient 0 Rejection Coefficient :0% rejection salts, solvents, buffers, etc.

8 ; 25% rejection molecules lower in molecular weight thanthe molecular weight cutoff of the membrane, but bigger than saltsFFiigguurree 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 usingdiafiltration isconcentration ofsamples on onesystem, minimizingthe risk of sampleloss orcontamination. 46 BioProcess InternationalMAY2003retentate volume and productconcentration do not change duringthe Diafiltration process. If water isused for diafiltering, salts will bewashed out and the retentateconductivity lowered.

9 If a Buffer is used for diafiltering,the new Buffer s 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 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 a 100% permeable solute can beremoved by washing through sixvolumes (6 DV) with the Buffer ofchoice.

10 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).