Chromatography

Enzyme preparations that have been clarified and concentrated are now in a suitable state for further purification by chromatography. For enzyme purification there are three principal types of chromatography utilising the ion-exchange, affinity and gel exclusion properties of the enzyme, usually in that order. Ion-exchange and affinity chromatographic methods can both rapidly handle large quantities of crude enzyme but ion-exchange materials are generally cheaper and, therefore, preferred at an earlier stage in the purification where the scale of operation is somewhat greater. Gel exclusion chromatography (also sometimes called 'gel filtration' or just 'gel chromatography' although it does not separate by a filtering mechanism, larger molecules passing more rapidly through the matrix than smaller molecules) is relatively slow and has the least capacity and resolution. It is generally left until last as an important final purification step and also as a method of changing the solution buffer before concentration, finishing and sale. Where sufficient information has been gathered regarding the size and variation of charge with pH of the required enzyme and its major contaminants, a rational purification scheme can be devised. A relatively quick analytical method for obtaining such data utilises a two-dimensional process whereby electrophoresis occurs in one direction and a range of pH is produced in the other; movement in the electric field determined by the size and sign of a protein's charge, which both depend on the pH. As the sample is applied across the range of pH, this method produces titration curves (i.e., charge versus pH) for all proteins present.

A large effort has been applied to the development of chromatographic matrices suitable for the separation of proteins. The main problem that has had to be overcome is that of ensuring the matrix has sufficiently large surface area available to molecules as large as proteins (i.e., they are macroporous) while remaining rigid and incompressible under rapid elution conditions. In addition, matrices must generally be hydrophilic and inert. Although the standard bead diameters of most of these matrices are non-uniform and fairly large (50 - 150 mm), many are now supplied as uniform-sized small beads (e.g., 4 - 6 mm diameter) which allows their use in very efficient separation processes (high performance liquid chromatography, HPLC), but at exponentially increasing cost with decreasing bead size. Relatively high pressures are needed to operate such columns necessitating specialised equipment and considerable additional expense. They are used only for the small-scale production of expensive enzymes, where a high degree of purity is required (e.g., restriction endonucleases and therapeutic enzymes).

Column manufacturers now supply equipment for monitoring and controlling chromatography systems so that it is possible to have automated apparatus which loads the sample, collects fractions and regenerates the column. Such equipment must, of course, have fail-safe devices to protect both column and product.