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作为蛋白A色谱的重要替代技术,混合模式色谱(mixed-mode chromatography,MMC)具有稳定、廉价和选择性好等优势。本文考察了以Sepharose Fast Flow为基质、4-(咪唑-1-基)苯胺(AN)为配基的新型色谱介质AN SepFF中配基密度对抗体吸附行为和分离性能的影响。研究结果表明,AN SepFF色谱介质在抗体吸附中具有21.2mmol/L的临界配基密度。在临界配基密度以上,抗体在色谱介质上具有较高的吸附容量和很低的解离常数并展现了相当程度的盐耐受性。动态吸附结果进一步表明,抗体的色谱介质中具有与商品化介质相当的动态吸附容量。抗体纯化的结果也表明,在临界配基浓度以上AN SepFF色谱表现出较好的纯化抗体的能力,其抗体产品纯度高于目前商品化的色谱介质;配基密度为55mmol/L时,AN SepFF色谱纯化抗体的收率达到91.9%。由此,AN SepFF色谱介质展现了巨大的应用前景。
As an important alternative to Protein A chromatography, mixed-mode chromatography (MMC) is stable, inexpensive and selective. In this paper, we investigated the effect of ligand density on the adsorption behavior and separation performance of AN SepFF with Sepharose Fast Flow as a substrate and 4- (imidazol-1-yl) aniline (AN) as ligand. The results show that the AN SepFF chromatographic medium has a critical ligand density of 21.2 mmol / L for antibody adsorption. Above the critical ligand density, the antibody has a higher adsorption capacity and a very low dissociation constant on the chromatographic medium and exhibits a considerable degree of salt tolerance. Dynamic adsorption results further indicate that the antibody has a dynamic adsorption capacity comparable to commercial media in the chromatographic medium. The results of antibody purification also showed that AN SepFF chromatography showed better antibody purification ability at the critical ligand concentration, and the antibody product purity was higher than that of the currently commercially available chromatographic medium. When the ligand density was 55 mmol / L, AN SepFF The yield of chromatographic purified antibody reached 91.9%. As a result, AN SepFF chromatographic media has shown tremendous promise.