Separation and purification of insulins on coated silica support functionalized with sialic acid by affinity chromatography

High performance liquid affinity chromatography (HPLAC) is a powerful method for purification and analysis of biological compounds. It combines the speed and the efficiency of high performance chromatography with the selectivity of affinity chromatography methods. The selectivity depends, first, on the nature of the ligand used; this latter must exhibit a specific binding affinity towards the product to purify; second, it depends on adsorption and desorption processus that improve, or not, the protein affinity for the ligand. The presence of N-acetyl neuraminic acid surrounding the insulin receptor structure shows that this acid may develop specific interactions with insulin. We performed the grafting of sialic acid on coated silica supports. The performances of these supports towards insulin were studied by HPLAC. The ligand specificity was tested in presence of two forms of insulin, porcine and bovine insulins, which, differ from each other by only two amino acids in their structure. The support carrying sialic acid exhibits both affinity and specificity for insulin in solution, since it allows the separation of the two forms of insulin. This result is confirmed by Reverse Phase High Performance Liquid Chromatography (RP-HPLC) assay. RP-HPLC is a widely used method for the separation of several compounds that exhibit structural homologies, such as, for instance and in particular, the different forms of insulin. In order to confirm the specificity and the affinity of the support bearing sialic acid for insulin, the elution of a pancreatic extract, consisting of numerous proteins including insulin, is performed. The support allows the insulin purification from a pancreatic extract with high purification yields.