Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 158

liquid chromatography). Fractionation of a single sample on such chromatographic columns typically
requires a minimum of several hours to complete. Low fl ow rates are required because, as the pro-
tein sample fl ows through the column, the proteins are brought into contact with the surface of the
chromatographic beads by direct (convective) fl ow. The protein molecules then rely entirely upon
molecular diffusion to enter the porous gel beads. This is a slow process, especially when compared
with the direct transfer of proteins past the outside surface of the gel beads by liquid fl ow. If a fl ow
rate signifi cantly higher than the diffusional rate is used, then 'protein band spreading' (and hence
loss of resolution) will result. This occurs because any protein molecules that have not entered the
bead will fl ow through the column at a faster rate than the (identical) molecules that have entered into
the bead particles. Such high fl ow rates will also result in a lowering of adsorption capacity, as many
molecules will not have the opportunity to diffuse into the beads as they pass through the column.
One approach that allows increased chromatographic fl ow rates without loss of resolution en-
tails the use of microparticulate stationary-phase media of very narrow diameter. This effectively
reduces the time required for molecules to diffuse in and out of the porous particles. Any reduc-
tion in particle diameter dramatically increases the pressure required to maintain a given fl ow rate.
Such high fl ow rates may be achieved by utilizing high-pressure liquid chromatographic systems.
By employing such methods, sample fractionation times may be reduced from hours to minutes.
The successful application of HPLC was made possible largely by (a) the development of pump
systems that can provide constant fl ow rates at high pressure and (b) the identifi cation of sui t-
able pressure-resistant chromatographic media. Traditional soft gel media utilized in low-pressure
applications are totally unsuited to high-pressure systems due to their compressibility.
In the context of protein purifi cation/characterization, HPLC may be used for analytical or prepara-
tive purposes. Most analytical HPLC columns available have diameters ranging from 4 to 4.6 mm and
lengths ranging from 10 to 30 cm. Preparative HPLC columns currently available have much wider
diameters, typically up to 80 cm, and can be longer than 1 m ( Figure 6.18). Various chemical groups
may be incorporated into the matrix beads; thus, techniques such as ion-exchange, gel-fi ltration, affi n-
ity, hydrophobic interaction and reverse-phase chromatography are all applicable to HPLC.
Many small proteins, in particular those that function extracellularly (e.g. insulin, GH and various
cytokines) are quite s table and may be fractionated on a variety of HPLC columns without signifi cant
denaturation or decrease in bioactivity. Preparative HPLC is used in industrial-scale purifi cation of
insulin and of IL2. In contrast, many larger proteins (e.g. blood factor VIII) are relatively labile, and
loss of activity due to protein denaturation may be observed upon high-pressure fractionation.
At both preparative and analytical levels, HPLC exhibits several important advantages com-
pared with low-pressure chromatographic techniques:

• HPLC offers superior resolution due to the reduction in bead particle size. The diffusional dis-

tance inside the matrix particles is minimized, resulting in sharper peaks than those obtained
when low-pressure systems are employed.

• Owing to increased fl ow rates, HPLC systems also offer much improved fractionation speeds,

typically in the order of minutes rather than hours.

• HPLC is amenable to a high degree of automation.

The major disadvantages associated with HPLC include cost and, to a lesser extent, capacity.
Thus, for both technical and economic reasons, preparative HPLC is employed almost exclusively