Biomedical Engineering Reference
In-Depth Information
6.5 Initialproductconcentration
The next phase of downstream processing usually entails concentration of the crude protein
product. This yields smaller product volumes, which are more convenient to work with and
can be subsequently processed with greater speed. Concentration may be achieved by inducing
product precipitation using salts, such as ammonium sulfate, or solvents, such as ethanol. The
precipitate is then collected (usually by centrifugation, but potentially also by fi ltration) and
the precipitate is redissolved in a small volume of processing buffer. Ion-exchange chroma-
tography (in which proteins bind to charged beads immobilized in a column) can also poten-
tially be used to concentrate protein solutions. This is discussed later in this chapter. Both of
these methods also result in limited protein purifi cation, as not all protein types present in the
crude preparation will co-precipitate or bind to the ion exchanger along with the target protein.
Ultrafi ltration, however, is by far the most common method now used to achieve initial product
concentration.
6.5.1 Ultrafi ltration
As discussed previously, the technique of microfi ltration is effectively utilized to remove whole
cells or cell debris from solution. Membrane fi lters employed in the microfi ltration process gener-
ally have pore diameters ranging from 0.1 to 10 µm. Such pores, while retaining whole cells and
large particulate matter, fail to retain most macromolecular components, such as proteins. In the
case of ultrafi ltration membranes, pore diameters normally range from 1 to 20 nm. These pores
are suffi ciently small to retain proteins of low molecular mass. Ultrafi ltration 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, ultrafi lters have been manufactured from cellulose acetate or cellulose nitrate.
Several other materials, such as polyvinyl chloride and polycarbonate, are now also used in mem-
brane manufacture. Such plastic-type membranes exhibit enhanced chemical and physical stabil-
ity when compared with cellulose-based ultrafi ltration membranes. An important prerequisite in
manufacturing ultrafi lters is that the material utilized exhibits low protein adsorptive properties.
Ultrafi ltration is generally carried out on a laboratory scale using a stirred-cell system
( Figure 6.6a). The fl at 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 fl ow through the ultrafi lter. Molecules of lower molecular mass than
the fi lter cut-off pore size (e.g. water, salt and low molecular weight compounds) all pass through
the ultrafi lter, thus concentrating the molecular species present whose molecular mass is signifi -
cantly 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 fl ow rate. Additional ultra-
fi lter formats used on a laboratory scale include cartridge systems, within which the ultrafi ltration
membrane is present in a highly folded format. In such cases, the pressure required to maintain a
satisfactory fl ow rate through the membrane is usually generated by a peristaltic pump.
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