巯基吡啶配基液相色谱固定相的制备及其对质粒的快速纯化

在基因治疗中,质粒是一种常用的非病毒型载体。由于目前市场上对质粒的需求量很大,因此有必要开发大规模的质粒制备技术。该文以双孔型流通色谱介质为基质,以巯基吡啶为配基,制得了液相色谱固定相,并考察了其对质粒DNA的纯化效果,分析了分离机理。结果表明:该分离介质对质粒的纯化是基于疏水作用;当上样量为10 mL (质粒质量浓度为0.30 mg/mL)、流速高达4 mL/min时,质粒仍然能以100%的收率实现纯化,其纯度为100%。该色谱介质对RNA的柱容量高达2.2 mg/mL。

Preparation of Biporous Polymeric Microspheres Coupled with Mercaptopyridine and Its Application in Rapid Purification of Plasmid DNA

Plasmid is a kind of popular nonviral vector in gene therapy.To produce large quantities of plasmid,an efficient large-scale purification process needs to be developed.In general,liquid chromatography is the most important tool for the analytical and preparative separation of plasmid DNA.In this study,biporous polymeric microspheres coupled with mercaptopyridine were synthesized for the purification of plasmid DNA.From the results of experiments,it can be inferred that the mechanism for the purification of the plasmid DNA with MP-BiPR is based on the hydrophobic interaction.When the load was 10 mL with the content of plasmid of 0.30 mg/mL and the content of RNA of 0.44 mg/mL,a flow rate was set up to 4 mL/min(20 cm/min).The separation was finished in 10 min,and the plasmid was completely recovered with undetectable impurities of nucleic acids and proteins.The load of the column for RNA was up to 2.2 mg/mL.