Phenomena of insulin peak fronting in size exclusion chromatography and strategies to reduce fronting

Insulin peak fronting in size exclusion chromatography (SEC) results in more than 10% yield loss in the production of insulin. The goal of this study is to understand the mechanisms of peak fronting and to develop strategies to reduce fronting and increase insulin yield. Chromatography experiments ruled out pressure surge, viscous fingering, and adsorption as the cause for peak fronting. Theoretical analysis based on a general rate model indicated that reversible dimerization is the major cause for peak fronting and reducing the dimerization equilibrium constant is the most effective method for reducing fronting. Two strategies were developed and tested to reduce the degree of dimer formation. The first strategy was to use 0.1N acetic acid as the presaturant and eluent. The second strategy was to use 0.8 or 2.8N acetic acid in 20vol.% denatured ethanol as the mobile phase. The experimental results showed that both strategies can reduce insulin peak fronting in SEC, maintain desired product purity, and increase insulin yield to higher than 98%.