Thesis final - after defense-7 - Jacobs University

Chapter 3
The correlation coefficients (r 2 ) of ASH with retention of proteins were 0.93, 0.83 and 0.96
for Toyopearl-Phenyl, Sepharose-Phenyl and Source-Phenyl, respectively. The correlation
coefficient reported in the Sepharose-Phenyl case was less than Toyopearl-Phenyl and
Source-Phenyl; the reason could be the heterogeneous distribution of the hydrophobic
hotspots on the base support. This kind of behavior was previously reported with
chromatographic separation of model proteins by Sepharose-Phenyl, where proteins of similar
ASH have exhibited different retention times (63).
In the previous reports, model proteins have been used to correlate ASH with the retention
behavior. This correlation was never examined as a function of the complex cell proteome (26,
27). The correlation of ASH with retention behavior was explored for the first time in this
work with the yeast cell proteome. The experimental data about salt concentrations and
corresponding hydrophobicity ranges at different elution stages can be exploited to predict the
separation of recombinant proteins employing yeast as a host cell proteome. The separation of
recombinant proteins by HIC can also be predicted in other host cell proteomes, once its ASH
is calculated. ASH was the only property which has revealed the trend lines to partially
differentiate among the base supports.
3.2.3.3.4. Other protein properties affecting the chromatographic behavior
The average flexibility was calculated for the tabulated proteins using the scale proposed by
Bhaskaran and Ponnuswamy (Tables 12-14) (98). The average flexibility has revealed a
statistically significant (r 2 = 0.80; p < 0.0001) direct correlation with the protein retention
during chromatography (Figure 27). The average flexibility has been reported before for its
direct relationship with the retention time of model proteins in HIC (62). This could be due to
the presence of cystein residues in a protein forming disulfide bridges. A protein with more
cystein content / and or disulfide bridges will be highly stable and less flexible towards
conformational changes. In contrast, proteins with less cystein residues will be highly flexible
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