UBC Theses and Dissertations
Custom isoelectric chromatofocusing : advanced models and methods for high-resolution protein purification Choy, Derek Yau Chung
Isoelectric chromatofocusing (ICF), a mode of chromatography by which proteins are separated based on changes in their charge with pH, is widely used at analytical scales, but its use in bio-product manufacturing has been limited. This is partly due to poor knowledge about operating ICF at scale, lack of understanding of its elution mechanisms, and the use of complex, costly buffers. Work presented in this thesis focuses on advancing ICF at both analytical and preparative scales. A method for generating pH gradients in ICF is developed using simple low-molecular-weight buffers. On anion and cation exchange media, linear gradients spanning more than six pH units are generated through isocratic or gradient interchange of loading and elution phases. The buffers used are selected to satisfy cost constraints and for compatibility with detection by UV absorption at 280 nm and mass spectrometry. A new surface-reaction/chemical-equilibria model is derived and solved by computer-aided simulations to predict pH and ionic strength profiles generated on anion and cation exchange columns. The model can be used for in silico design of custom-shaped elution profiles to improve separation performance. The method is used to achieve high purity and process throughput of a desired isoform of recombinant N-lobe of human transferrin produced by Pichia pastoris using custom isocratic ICF on preparative media. Gradient sculpting methods are used to enhance ICF as the first dimension in a multidimensional separation platform used for the detection and analysis of O-linked N-acetylglucosamine modified proteins within the proteome of differentiated C2C12 mouse myoblast cells. Finally, a model of protein transport and binding in ICF is developed and used to show that elution is not dictated solely by a protein’s isoelectric point (pI), but is instead multi-modal in nature with Donnan equilibria, ion-exchange, and ion-displacement effects at work. The model predicts how simultaneous modulation of ionic strength and pH during elution can greatly improve the separation of proteins with similar pI’s; elution characteristics including retention time, peak width and resolution can likewise be improved. By coupling mathematical relationships describing these elution mechanisms to the solution of the continuity equation, protein elution times are accurately predicted.
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