Double-coated silica supports for high-performance affinity chromatography of proteins

In order to prepare easily derivatizable supports for high-performance affinity chromatography (HPAC), the advantages of traditional polysaccharide-based supports were combined with the excellent mechanical properties of silica by coating the porous silica beads with a double layer of polysaccharide. The starting material was preliminarily impregnated with dextran or agarose, substituted with a calculated amount of positively charged diethylaminoethyl (DEAE) functions, in order to neutralize the cation-exchange capacity. These silica beads were then recoated by a second coupling with a native dextran or agarose so the DEAE functions introduced by the first coating could be overlayed and the coating state of the silica beads was further improved. The passivation of silica was confirmed by eluting standard proteins on the double-coated silica supports in high-performance size-exclusion chromatography. The elution of an acidic biopolymer, heparin, on different coated silica supports under gradient conditions demonstrated the major improvement of the native polymeric overlayer on the ionic properties of the support. These double-coated silica supports can also easily be activated by classical activation methods and coupled with active ligands (protein A and heparin). The active supports grafted by protein A were used in HPAC of human immunoglobulin G. The double-coated silica supports grafted by heparin were used in HPAC of human alpha-thrombin and human antithrombin III and for the purification of bovine thrombin from a commercial crude thrombin preparation.