Methods to reduce fronting and increase yield in batch size exclusion chromatography
Abstract
Peak fronting occurs in size exclusion chromatography, SEC, resulting in 10% loss in insulin yield. The goal of this project is to understand the mechanism of insulin peak fronting and develop a strategy to reduce fronting and increase insulin yield in SEC. Chromatography experiments ruled out pressure surge, viscous fingering, and adsorption as the cause for fronting. Theoretical analysis based on a general rate model shows that reversible dimerization can cause peak splitting, merging, tailing and fronting. Slow association and dissociation result in separated monomer and dimer peaks. Fast association and slow dissociation result in one dimer peak. Slow association and fast dissociation result in one monomer peak. Intermediate association and dissociation rates result in a merged dimerlmonomer peak with fronting if monomers dominate. A diagram based on the two relative rates is generated to predict general peak shape and retention behavior. The analysis shows that reducing the dimerization equilibrium constant is most effective for reducing fronting. An in-situ spectroturbidimetry method with a photodiode array detector is developed and tested for direct probing of insulin aggregation. In acetic acid concentrations from 0.1 N to 1 N at 25°C, the aggregation number, nm, ranges from 2.9 to 1.6. Insulin at 25°C in 3.5 N acetic acid or at 4°C in 2.8 N acetic acid with 20 vol% denatured ethanol is monomeric. In-situ probing of SEC effluents in 3.5 N acetic acid at 4°C shows nm = 1.6 at the fronting portion and nm = 1.1 at the tailing portion. Analytical HPLC results imply that the aggregates are reversible. Based on the theoretical analysis and the in-situ probing results with spectroturbidimetry, a strategy for reducing insulin fronting is formulated. The strategy is to replace the original mobile phase (1 N acetic acid) with 2.8 N acetic acid plus 20 vol% denatured ethanol. Experimental results for this strategy show that fronting is reduced, and insulin yield can be increased to 99%.
Degree
Ph.D.
Advisors
Wang, Purdue University.
Subject Area
Chemical engineering
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