Thesis final - after defense-7 - Jacobs University

Chapter 1
cell proteome to facilitate the separation of the target protein in the real bioprocess. Several
expert systems have been proposed to link the efficiency of a unit operation with the protein
properties and to separate the target protein from the other discrete number of contaminants.
Most of these approaches to understand the chromatographic behavior of proteins were
limited to the use of model proteins instead of protein mixtures in the host cell proteome. No
comprehensive struggle has been made before to define the contaminant proteome of real
recombinant host in order to investigate the individual contaminant behavior during its
chromatographic separation. A paradigm shift from focussing on protein product to defining
the major contaminant might give a more realistic picture and could also make this approach
more suitable. The characterization of host related proteins is also important to maintain the
product quality (9, 19).
Chromatography is an important unit operation of any downstream process which helps to
remove the contaminants during the purification of biological materials. Chromatographic
separation of protein mixtures is becoming one of the most effective and widely used means
of purifying individual proteins. The chromatographic methods for protein separations are
based on the general physicochemical properties of the proteins (Figure 1) (20). Minor
differences between various proteins such as size, charge and hydrophobicity can be used to
purify one protein from the other proteins during chromatography. Chromatographic
separations are usually based on protein characteristics such as charge (ion exchange
chromatography), size (size exclusion chromatography), isoelectric point (chromato focussing)
affinity (affinity chromatography) and hydrophobicity (hydrophobic interaction
chromatography and reverse phase chromatography). Hydrophobic interaction
chromatography (HIC) is a chromatographic method based on protein hydrophobicity (4).
HIC was considered as more significant than other chromatographic methods, due to its mild
hydrophobic nature and feasibility for the separation of less stable proteins (21). HIC is a
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