Thesis final - after defense-7 - Jacobs University

Chapter 1
proteins. It is this difference which can be exploited in HIC for protein separations. Protein
hydrophobicity is the main physicochemical property which has a decisive role in retention
during chromatography. There is no universally agreed single method to calculate protein
hydrophobicity, however there is a general agreement that it can be determined by the
hydrophobic contribution of the amino acid residues (25, 26). The operating conditions such
as mobile phase properties (salt type, ionic strength and pH), stationary phase characteristics
(chemical nature of the backbone, type of hydrophobic ligand and substitution level of the
resin) and temperature play important roles in HIC (27, 28). Hydrophobic interactions are the
most important non covalent forces which have the ability to maintain the native structures of
the proteins at high salts concentrations. The basic principle of the HIC is different from size
exclusion and ion exchange chromatography (IEC) and thus can be used for the products
which have no possibility to be separated by these techniques. HIC is preferred over other
chromatographic methods if proteins of similar size or isoelectric point coordinates have to be
separated from each other. HIC can also be used as an ideal step, after the separation of
biomaterials during IEC at high salt concentrations. Due to the entries of several new HIC
media and better understanding of the factors affecting the retention, HIC has become one of
the most widely used method for purification of proteins such as serum proteins, membrane
proteins, nuclear proteins, receptors and recombinant proteins for research and industrial
applications (14, 23, 29). The enzymes of commercial importance such as lipases, amylases,
streptokinases can be purified utilizing HIC (30). Several expert systems have been produced
to exploit the physicochemical properties of the proteins in order to separate the desired
product from other contaminants in HIC (31, 32). In most of these approaches, the prediction
of protein retention was limited to the use of model proteins instead of the host cell proteome
(33). Studies of the host cell proteome which produces the product of interest can help in the
design of the process; give in depth knowledge of the target and undesired proteins for the
efficient downstream processing. No large scale effort has been made to study the
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