Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 157

moieties of the hydroxyapatite matrix. Elution of bound species from such columns is normally
achieved by irrigation with a potassium phosphate gradient.
6.6.11 Chromatofocusing
Fractionation by chromatofocusing separates proteins on the basis of their isoelectric points. This
technique basically involves percolating a buffer of one pH through an ion-exchange column that
is pre-equilibrated at a different pH. Owing to the natural buffering capacity of the exchanger, a
continuous pH gradient may be set up along the length of the column. In order to achieve maxi-
mum resolution, a linear pH gradient must be constructed. This necessitates the use of an eluent
buffer and exchanger that exhibit even buffering capacity over a wide range of pH values. The
range of the pH gradient achieved will obviously depend on the pH at which the ion exchanger
is pre-equilibrated and the pH of the eluent buffer. The sample is applied, usually in the running
buffer, whose pH is lower than that of the pre-equilibrated column. After sample application, the
column is constantly percolated with a specially formulated buffer that establishes an increasing
pH gradient down the length of the column.
Upon sample application, negatively charged proteins immediately adsorb to the anion exchanger,
while positively charged proteins fl ow down the column. Owing to the increasing pH gradient formed,
such positively charged proteins will eventually reach a point within the column where the column
pH equals their own pI values (their isoelectric points, i.e. the pH value at which the protein has an
overall net charge of zero). Immediately upon further migration down the column such proteins
become negatively charged, as the surrounding pH values increase above their pI values; hence,
they bind to the column. Overall, therefore, upon initial application of the elution buffer, all protein
species will migrate down the column until they reach a point where the column pH is marginally
above their isoelectric points. At this stage they bind to the anion exchanger. Proteins of differing
isoelectric points are thus fractionated on the basis of this parameter of molecular distinction.
The pH gradient formed is not a static one. As more elution buffer is applied, the pH value at any
given point along the column is continually increasing. Thus, any protein that binds to the column
will be almost immediately desorbed, as once again it experiences a surrounding pH value above
its pI and becomes positively charged. Any such desorbed protein fl ows down the column until it
reaches a further point where the pH value is marginally above its pI value, and it again rebinds. This
process is repeated until the protein emerges from the column at its isoelectric point. To achieve the
best results, the isoelectric point (pI value) of the required protein should ideally be in the middle
of the pH gradient generated. Chromatofocusing can result in a high degree of protein resolution,
with protein bands being eluted as tight peaks. This technique is particularly effective when used
in conjunction with other chromatographic methods during protein purifi cation. Most documented
applications of this method still pertain to laboratory-scale procedures. Scaling up to industrial level
is somewhat discouraged by economic factors, most notably the cost of the eluent required.
6.7 High-performance liquid chromatography of proteins
Most of the chromatographic techniques described thus far are usually performed under relatively low
pressures, where fl ow rates through the column are generated by low-pressure pumps (i.e. low-pressure