Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 146

Column containing
chromatographic beads

Low molecular
weight substance

Low mol. wt. substance

A (Protein fraction)

280

Low mol. wt.
substance has
entered the
beads Protein

Sample to be
applied to column

(a)

Fraction no.

(c)

Fractions

(b)

Figure 6.9 The application of gel-fi ltration chromatography to separate proteins from molecules of much
lower molecular weight. The mobile phase (the 'running buffer') will be devoid of the molecular species to be
removed from the protein. Highly cross-linked porous beads are used, which exclude all protein molecules. The
lower molecular weight substances, however, can enter the beads; therefore, their progress down through the
column will be retarded (a and b). The earlier fractions collected will contain the proteins, and the latter frac-
tions will contain the low molecular weight contaminants (c). In practice, this 'group separations' application
of gel-fi ltration chromatography is mainly used to separate proteins from salt (e.g. af ter an ammonium sulfate
precipitation step) or for buffer exchange. Note : in practice, the chromatographic beads are tightly packed
in the column. They are separated from each other in this diagram only for the purpose of clarity. Also, the
drawing is not to scale; protein molecules are considerably smaller than individual beads

side groups, whereas lysine, arginine and histidine have positively charged basic side groups
( Figure 6.10). Protein molecules, therefore, possess both positive and negative charges, largely
due to the presence of varying amounts of these seven amino acids. (N-terminal amino groups
and the C-terminal carboxy groups also contribute to overall protein charge characteristics.)
The net charge exhibited by any protein depends on the relative quantities of these amino acids
present in the protein, and on the pH of the protein solution. The pH value at which a protein
molecule possesses zero overall charge is termed its isoelectric point (pI). At pH values above
its pI, a protein will exhibit a net negative charge, whereas proteins will exhibit a net positive
charge at pH values below the pI.
Ion-exchange chromatography is based upon the principle of reversible electrostatic attrac-
tion of a charged molecule to a solid matrix that contains covalently attached side groups of
opposite charge ( Figure 6.11). Proteins may subsequently be eluted by altering the pH or by
increasing the salt concentration of the irrigating buffer. Ion-exchange matrices that contain
covalently attached positive groups are termed anion exchangers. These will adsorb anionic
proteins, e.g. proteins with a net negative charge. Matrices to which negatively charged groups
are covalently attached are termed cation exchangers, adsorbing cationic proteins, e.g. posi-
tively charged proteins. Positively charged functional groups (anion exchangers) include spe-
cies such as aminoethyl and diethylaminoethyl groups. Negatively charged groups attached
to sui table matrices forming cation exchangers include sulfo- and carboxy-methyl groups
( Table 6.3).