Design of high affinity low molecular weight displacers and investigation of the selectivity of displacers and proteins in ion exchange systems.

摘要

This thesis seeks to address several key issues regarding the selectivity of proteins and displacers in ion exchange systems. Finding the correct operating conditions that maximize the selectivity of chromatographic purification systems is one of the biggest challenges in biopurification. Secondary interactions that are present in cation and anion exchange resins have been studied by using homologous series of molecules in order to establish rules of thumb for the design of high affinity low molecular weight displacers. Some important findings from the studies on anion exchange materials suggested that successful displacer molecules should possess a high degree of aromaticity, specifically from benzene rings.; Another method for creating selectivity changes in ion exchange resins is changing the mobile phase salt type. A large number of proteins were studied for their retention on different stationary phases under linear gradient conditions in the presence of different salt counter-ions. The salt effects are classified as non-specific and specific. The former is a generic effect on all proteins which is observed as a decrease in retention of proteins as the displacing salt strength is increased. For example, retention times are generally longer in NaCl compared to Na₂SO₄ when a constant gradient is maintained. Specific effects are indicated by changes in the elution order of proteins. The effect of displacer chemistry and salt type on displacer selectivity has also been studied by characterizing a displacer's efficacy in the presence of model proteins. The information obtained from changes in protein and displacer selectivity is then utilized to create selective elution techniques for protein purification. It was observed that not only the displacer selectivity, but also the multi-component adsorption effects impact the purification profile and efficiency.; Finally, an attempt has been made to develop a technique to understand fundamentals of ion-ion and displacer-ion interactions through molecular dynamic simulations. Simple systems of salt counter-ions and displacer molecules were used to validate the effectiveness of MD simulations for characterization of selectivity in ion exchange systems with promising results.; The application of displacement purification has been extended beyond its traditional applications for the purification of anti-sense oligonucleotides with different modifications as well as very high molecular weight proteins. Successful separations were achieved by utilizing low molecular weight displacers on ion exchange systems.; The results of this research shed significant light on understanding and characterizing the sources of selectivity in ion exchange systems. Importantly, it is now established that displacement chromatography is a powerful tool which can be tailored to achieve very difficult separations once the underlying factors, including displacer chemistry and salt type, governing selectivity are understood.