High-performance affinity chromatography of insulin on coated silica grafted with sialic acid.

Abstract

High-performance liquid affinity chromatography (HPLAC) is a powerful method for the purification of biological compounds, owing to its specificity, speed and high resolution. We developed new chromatographic supports based on porous silica beads. In order to minimize non-specific interaction between the silanol groups at the silica surface and biological molecules, the beads are coated with dextran carrying a calculated amount of positively charged functions. Such supports have the mechanical properties of the starting inorganic material. Moreover, they can be easily activated and functionalized by active ligands using conventional coupling methods. In the present study, N-acetylneuraminic acid (NANA), a member of the sialic acid family, is coupled to dextran coated silica beads to obtain affinity supports. This class of compounds seems to play an important role in the cell recognition mechanism. In particular, sialic acids are present in the structure of the cellular receptors for insulin. By HPLAC, we can study the interactions between coated silica grafted with NANA and insulin. It is also possible to use these active supports to purify the compounds by affinity chromatography. However, it is important to determine and optimize the conditions for adsorption and desorption of insulin on supports grafted with sialic acid and to estimate the chromatographic performances of these active phases.