Abstract
We developed the synthesis of new supports for the purification of insulin and IgG by affinity chromatography. The preparation of such an affinity support is performed in two steps. First, silica beads are coated with dextran polymers carrying a calculated amount of positively charged diethylaminoethyl groups in order to mask negative charges at its surface. Second, ligand is immobilized using a coupling agent. This support combines the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. The existence of N-acetylneuraminic acid (sialic acid) in insulin receptor and in the antigenic determinant of IgG suggests that such an acid may develop specific interactions usable in affinity chromatography. Therefore, N-acetylneuraminic acid was used as an active ligand. The immobilization of sialic acid can be carried out by using the conventional coupling agent: the carbonyldiimidazole. The performances of these supports grafted by sialic acid were studied by high-performance liquid affinity chromatography (HPAC). The optimization of the chromatographic conditions (support characteristics and mobile phase) enabled us to observe a behavior of the type “thiophilic” of the support, which does not contain sulfone group. This new affinity support allowed a one-step separation of the IgG from mouse ascitic fluids and also allowed the insulin purification from a pancreatic extract with a good purification yields.
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