Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 144

6.6.1 Size-exclusion chromatography (gel fi ltration)
Size-exclusion chromatography, also termed gel-permeation or gel-fi ltration chromatography, sep-
arates proteins on the basis of their size and shape. As most proteins fractionated by this technique
are considered to have approximately similar molecular shape, separation is often described as
being on the basis of molecular mass, although such a description is somewhat simplistic.
Fractionation of proteins by size-exclusion chromatography is achieved by percolating the protein-
containing solution through a column packed with a porous gel matrix in bead form ( Figure 6.8). As
the sample travels down the column, large proteins cannot enter the gel beads and hence are quickly
eluted. The progress of smaller proteins through the column is retarded, as such molecules are capa-
ble of entering the gel beads. The internal structure of the matrix beads could be visualized as a maze,
through which proteins small enough to enter the gel must pass. Various possible routes through this
maze are of varied distances. All proteins capable of entering the gel are thus not retained within the
gel matrix for equal time periods. The smaller the protein, the more potential internal routes open to
it and, thus, generally, the longer it is retained within the bead structure. Protein molecules, therefore,
are usually eluted from a gel-fi ltration column in order of decreasing molecular size.
In most cases the gel matrices utilized are prepared by chemically cross-linking polymeric mol-
ecules such as dextran, agarose, acrylamide and vinyl polymers. The degree of cross-linking con-
trols the average pore size of the gel prepared. Most gels synthesized from any one polymer type
are thus available in a variety of pore sizes. The higher the degree of cross-linking introduced,
the smaller the average pore size and the more rigid the resultant gel bead. Highly cross-linked
gel matrices have pore sizes that exclude all proteins from entering the gel matrix. Such gels may
be used to separate proteins from other molecules that are orders of magnitude smaller, and are
often used to remove low molecular weight buffer components and salts from protein solutions
( Figure 6.9).
Size-exclusion chromatography is rarely employed during the initial stages of protein purifi ca-
tion. Small sample volumes must be applied to the column in order to achieve effective resolution.
Application volumes are usually in the range of 2-5 per cent of the column volume. Furthermore,
columns are easily fouled by a variety of sample impurities. Size-exclusion chromatography is
thus often employed towards the end of a purifi cation sequence, when the protein of interest is
already relatively pure and is present in a small, concentrated volume. After sample application,
the protein components are progressively eluted from the column by fl ushing with an appropri-
ate buffer. In many cases, the eluate from the column passes through a detector. This facilitates
immediate detection of protein-containing bands as they elute from the column. The eluate is
normally collected as a series of fractions. On a preparative scale, each fraction may be a number
of litres in volume. Although size-exclusion chromatography is an effective fractionation tech-
nique, it generally results in a signifi cant dilution of the protein solution relative to the starting
volume applied to the column. Column fl ow rates are also often considerably lower than fl ow rates
employed with other chromatographic media. This results in long processing times, which, for
industrial applications, has adverse process cost implications.
6.6.2 Ion-exchangechromatography
Several of the 20 amino acids that constitute the building blocks of proteins exhibit charged
side chains. At pH 7.0, aspartic and glutamic acids have overall negatively charged acidic