Ultrafiltration

As discussed previously, the technique of microfiltration is effectively utilized to remove whole cells or cell debris from solution. Membrane filters employed in the microfiltration process generally have pore diameters ranging from 0.1 to 10 ^m. Such pores, while retaining whole cells and large particulate matter, fail to retain most macromolecular components, such as proteins. In the case of ultrafiltration membranes, pore diameters normally range from 1 to 20 nm. These pores are sufficiently small to retain proteins of low molecular mass. Ultrafiltration membranes with molecular mass cut-off points ranging from 1 to 300 kDa are commercially available. Membranes with molecular mass cut-off points of 3, 10, 30, 50, and 100 kDa are most commonly used.

Traditionally, ultrafilters have been manufactured from cellulose acetate or cellulose nitrate. Several other materials, such as polyvinyl chloride and polycarbonate, are now also used in membrane manufacture. Such plastic-type membranes exhibit enhanced chemical and physical stability when compared with cellulose-based ultrafiltration membranes. An important prerequisite in manufacturing ultrafilters is that the material utilized exhibits low protein adsorptive properties.

Ultrafiltration is generally carried out on a laboratory scale using a stirred-cell system (Figure 6.6a). The flat membrane is placed on a supporting mesh at the bottom of the cell chamber and the material to be concentrated is then transferred into the cell. Application of pressure, usually nitrogen gas, ensures adequate flow through the ultrafilter. Molecules of lower molecular mass than the filter cut-off pore size (e.g. water, salt and low molecular weight compounds) all pass through the ultrafilter, thus concentrating the molecular species present whose molecular mass is significantly greater than the molecular mass cut-off point. Concentration polarization (the build-up of a concentrated layer of molecules directly over the membrane surface that are unable to pass through the membrane) is minimized by a stirring mechanism operating close to the membrane surface. If unchecked, concentration polarization would result in a lowering of the flow rate. Additional ultrafilter formats used on a laboratory scale include cartridge systems, within which the ultrafiltration membrane is present in a highly folded format. In such cases, the pressure required to maintain a satisfactory flow rate through the membrane is usually generated by a peristaltic pump.

N2 under pressure "

Protein molecule-

Lower molecular,/]" weight molecule y

Effluent

Ultrafilter

Porous support for filter

Ultrafilter

Porous support for filter

Effluent

Figure 6.6 Ultrafiltration separates molecules based on size and shape. (a) Diagrammatic representation of a typical laboratory-scale ultrafiltration system. The sample (e.g. crude protein solution) is placed in the ultrafiltration chamber, where it sits directly above the ultrafilter membrane. The membrane, in turn, sits on a macroporous support to provide it with mechanical strength. Pressure is then applied (usually in the form of an inert gas), as shown. Molecules larger than the pore diameter (e.g. large proteins) are retained on the upstream side of the ultrafilter membrane. However, smaller molecules (particularly water molecules) are easily forced through the pores, thus effectively concentrating the protein solution (see also (b)). Membranes that display different pore sizes, i.e. have different molecular mass cut-off points, can be manufactured. (c) Photographic representation of an industrial-scale ultrafiltration system (photograph courtesy of Elga Ltd, UK)

Figure 6.6 Ultrafiltration separates molecules based on size and shape. (a) Diagrammatic representation of a typical laboratory-scale ultrafiltration system. The sample (e.g. crude protein solution) is placed in the ultrafiltration chamber, where it sits directly above the ultrafilter membrane. The membrane, in turn, sits on a macroporous support to provide it with mechanical strength. Pressure is then applied (usually in the form of an inert gas), as shown. Molecules larger than the pore diameter (e.g. large proteins) are retained on the upstream side of the ultrafilter membrane. However, smaller molecules (particularly water molecules) are easily forced through the pores, thus effectively concentrating the protein solution (see also (b)). Membranes that display different pore sizes, i.e. have different molecular mass cut-off points, can be manufactured. (c) Photographic representation of an industrial-scale ultrafiltration system (photograph courtesy of Elga Ltd, UK)

Large-scale ultrafiltration systems invariably employ cartridge-type filters (Figure 6.6c). This allows a large filtration surface area to be accommodated in a compact area. Concentration polarization is avoided by allowing the incoming liquid to flow across the membrane surface at right angles, i.e. tangential flow. The ultrafiltration membrane may be pleated, with subsequent joining of the two ends to form a cylindrical cartridge. Alternatively, the membrane may be laid on a spacer mesh and this may then be wrapped spirally around a central collection tube, into which the filtrate can flow.

Another widely used membrane configuration is that of the hollow fibres. In this case, the hollow cylindrical cartridge casing is loaded with bundles of hollow fibres. Hollow fibres have an outward appearance somewhat similar to a drinking straw, although their internal diameters may be considerably smaller. In this configuration, the liquid to be filtered is pumped through the central core of the hollow fibres. Molecules of lower molecular mass than the membrane rated cut-off point pass through the walls of the hollow fibre. The permeate, which emerges from the hollow fibres along all of their length, is drained from the cartridge via a valve. The concentrate emerges from the other end of the hollow fibre and is collected by an outlet pipe; this is referred to as the retentate. The permeate is then normally discarded, whereas the retentate, containing the protein of interest, is processed further. The retentate may be recycled through the system if further concentration is required.

Ultrafiltration has become prominent as a method of protein concentration for a variety of reasons:

• the method is very gentle, having little adverse effect on bioactivity of the protein molecules;

• high recovery rates are usually recorded, with some manufacturers claiming recoveries of over 99 per cent;

• processing times are rapid when compared with alternative methods of concentration;

• little ancillary equipment is required.

One drawback relating to this filtration technique is its susceptibility to rapid membrane clogging. Viscous solutions also lead to rapid decreases in flow rates and prolonged processing times.

Ultrafiltration may also be utilized to achieve a number of other objectives. As discussed above, it may yield a limited degree of protein purification and may also be effective in depyrogenating solutions. This will be discussed further in Chapter 7. The technique is also widely used to remove low molecular mass molecules from protein solutions by diafiltration.

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