Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 142

per cent of all molecules that freely cross the membrane will have been removed ('fl ushed out')
from the solution. Removal of low molecular weight contaminants from protein solutions may also
be achieved by other techniques, such as dialysis or gel-fi ltration chromatography. Diafi ltration,
however, is emerging as the method of choice, as it is quick, effi cient and utilizes the same equip-
ment as used in ultrafi ltration.
6.6 Chromatographicpurifi cation
Once the protein is recovered from its producer source and concentrated it must be purifi ed to ho-
mogeneity. In other words, all contaminant proteins and other potential contaminants of potential
medical signifi cance (discussed in Chapter 7) must be removed. Purifi cation is generally achieved
by column chromatography.
Column chromatography refers to the separation of different protein types from each other
according to their differential partitioning between two phases: a solid stationary phase (the
chromatographic beads, usually packed into a cylindrical column) and a mobile phase (usu-
ally a buffer). With the exception of gel fi ltration, all forms of chromatography used in protein
purifi cation protocols are adsorptive in nature. The protein mix is applied to the column (usu-
ally) under conditions that promote selective retention of the target protein. Ideally, this target
protein should be the only one retained on the column, but this is rarely attained in practice.
After sample application, the column is washed ('irrigated') with mobile phase in order to
fl ush out all unbound material. The composition of the mobile phase is then altered in order to
promote desorption of the bound protein. Fractions of eluate are collected in test tubes, which
are then assayed for both total protein and for the protein of interest ( Figure 6.7). The fractions
containing the target protein are then pooled and subjected to the next step in the purifi cation
process.
Individual protein types possess a variety of characteristics that distinguish them from other
protein molecules. Such characteristics include size and shape, overall charge, the presence of
surface hydrophobic groups and the ability to bind various ligands. Quite a number of protein mol-
ecules may be similar to one another if compared on the basis of any one such characteristic. All
protein types, however, present their own unique combination of characteristics, a protein chro-
matographic 'fi ngerprint'. Various chromatographic techniques have been developed that separate
proteins from each other on the basis of differences in such characteristics ( Table 6.2). Utilization
of any one of these methods to exploit the molecular distinctiveness usually results in a dramatic
increase in the purity of the protein of interest. A combination of methods may be employed to
yield highly purifi ed protein preparations.
In general, a combination of two to four different chromatographic techniques is employed in
a typical downstream processing procedure. Gel-fi ltration and ion-exchange chromatography are
amongst the most common. Affi nity chromatography is employed wherever possible, as its high
biospecifi city facilitates the achievement of a very high degree of purifi cation. Examples include
the use of immunoaffi nity chromatography to purify blood factor VIII and lysine affi nity chroma-
tography to purify tPA.
As with most aspects of downstream processing, the operation of chromatographic systems is
highly automated and is usually computer controlled. Whereas medium-sized process-scale chro-
matographic columns (e.g. 5-15 l capacity) are manufactured from toughened glass or plastic, larger