| Hydrophobic interaction chromatography (HIC) is gaining popularity for purification of biologics because it accomplishes separation based on a different set of molecular characteristics to more popular methods such as ion-exchange chromatography. The existing retention models for HIC account for the effect of salt, but the effects of adsorbent and protein properties are not completely understood. In addition, very few studies have been performed to study protein transport in HIC although this area is important to process design and scale-up. The goals of this thesis were to characterize the adsorbents used in HIC and to determine adsorption and transport properties necessary for designing HIC processes.; First, inverse size exclusion chromatography was used to determine the key physical properties, specifically the pore size distributions, of a set of HIC adsorbents. These were then used to explain subsequently measured adsorption and transport trends.; Isocratic elution experiments showed a very clear trend in that protein molecular mass and structural stability affect retention, while protein recovery is sensitive to structural stability and especially the nature of the adsorbent base matrix.{09}A general thermodynamic relation was derived that predicts that protein retention in HIC increases under conditions that decrease protein solubility. This correlation is consistent with the experimental results that solubility, in terms of its surrogate the second osmotic virial coefficient, correlates well with HIC retention in many cases, including correctly predicting the reverse Hofmeister effects. However, solubility could not explain retention behavior under some conditions. In those cases, protein-surface interactions or conformational change could be important determinants of protein adsorption.; The adsorption isotherms of proteins in HIC were relatively "soft", and well-defined plateau regions were not observed. The static capacities of the media varied depending on salt concentration and the protein and adsorbent types. The protein accessible surface area appears to be the main factor determining the binding capacity. The dynamic capacities of the adsorbents were 10--70% of the static capacities, depending on adsorbent particle size and feed flow rate.; The effective pore diffusivities of model proteins in HIC media were determined using the general linear rate model. The pore diffusivities obtained from this method are generally accurate for proteins with low structural flexibility but not for more flexible ones, presumably because conformational change effects contribute significantly to the overall rate limitations. (Abstract shortened by UMI.)... |
