Effect of pH, Temperature and Protein Tertiary Structure on Biomolecular Separation Engineering in Ion-Exchange Chromatography

Abstract Bioseparation technology remains a key bottleneck in biomolecular manufacturing. Several important unit operations dominate the industry, with ion-exchange chromatography leading the way in providing a scalable, robust and cost-effective way to isolate a desired biomolecule. Engineering of these separation technologies remains a major hurdle, as methods that were originally developed by analytical chemistry are employed to describe the unit operations. In this thesis, the primary goal is to provide a solid engineering analysis of the ion-exchange chromatography system to aid the practicing engineer in modeling and scale-up.The other major goal of this work is to provide sufficient explanation as to what is the effect of operating variables such as pH and temperature on the separation. When ion-exchange chromatography is used for biomolecular separation, pH is the key variable that provides the conditions for the biomolecule to have a sufficient charge to get adsorbed onto the adsorbent material. Modeling of the binding force was achieved by measuring the equilibrium constant KA for the biomolecule, and then correlating the values to the net charges of the biomolecules at the pH conditions. In anion-exchange chromatography, more basic pH led to higher retention due to the higher negative charge of the biomolecules. More acidic pH led to more positive ionization of the biomolecules and contributed to higher retention.For the case of temperature, industrial guidelines do not provide any direction as to how changing the temperature affects the separation. In anion-exchange, higher retention and resolution was observed with increasing temperature, and in cation-exchange, higher retention and resolution were observed with decreasing temperature. These effects are completely opposite from one another and can provide a basis for new modes of temperature driven ion-exchange separation. Using the retention data for different temperatures, an Arrhenius plot was created to explain the trends that were observed. For the cation-exchange chromatography, a negative activation energy was observed and for anion-exchange chromatography a positive activation energy was observed.The tertiary structure of the protein can also provide very useful information about the nature of the binding affinity of the biomolecule to the adsorbent ligand. The surface charges of the biomolecule appear to be instrumental in adsorption, providing for a higher retention when compared to a homologous biomolecule without the same surface charges.Taking these established effects for the chemical nature of the process, a comprehensive model for the ion-exchange biomolecular separation was established. In the model, physical variables were investigated first to determine the extent by which they affect the separation. Afterwards, the correlations were coupled to the trends derived from the experimental data so that the elution chromatograms at any pH and temperature for ion-exchange chromatography can be computed in a rapid and useful fashion.This work establishes a good theoretical basis for analysis of packed bed sorption processes to investigate the effect of physical variables such as pH, temperature and the tertiary structure. The data and models presented here provide a practicing engineer or researcher good theoretical framework for understanding the process, which can then be used in a predictive way for scale-up of the process.