Selective non-adsorption preparative chromatography of proteins
Abstract
A novel approach to protein purification is developed and evaluated where the protein of interest is allowed to pass through a series of chromatographic columns while contaminating proteins are retained. This approach has the combined advantages of speed, economy, scalability and gentleness to the purified proteins. Use of combined anion and cation exchange sorbents at a protein's isoelectric point and low salt concentrations results in a situation where the protein is not retained while many others are. This combination was used in the purification of IgGl from bovine serum to 95% purity in a single step. The preparative aspects of this purification were investigated, including frontal loading, scalability, continuous processing and effect of increasing mobile phase flow. The potential of this ion exchange combination for other proteins was evaluated and the technique for selecting proper mobile phase conditions for any protein was developed. Additional separation modes developed for this non-adsorption approach were size exclusion and affinity chromatography. A series of varying high ligand density sorbents was synthesized. These sorbents allow the frontal purification of proteins via their hydrophobic interaction at moderate ionic strength. A series of these columns was used to fractionate bovine serum. Large pore polystyrene sorbents were evaluated for the rapid separation of proteins. It was demonstrated that these sorbents allow the mobile phase to flow through them at high flow rate. This allows the sorbents to bind more protein at higher flow rates rather than less. The preparative application of this phenomenom was investigated.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Biochemistry
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