重组人脑利钠肽制备中内毒素去除工艺研究

目的]在重组人脑利钠肽(BNP)的分离纯化过程中去除内毒素。方法]选用Chelating Sepharose FF亲和柱层析、Q Sepharose FF阴离子交换柱层析、Source 15反相柱层析、SP Sepharose FF阳离子交换柱层析组合的方法对BNP进行分离纯化；按照2010版药典中鲎试剂法检测各层析柱纯化阶段得到蛋白溶液中的内毒素含量；并用HPLC方法检测各阶段所得蛋白的浓度和纯度。结果]BNP制备过程中每一步层析在内毒素去除方面都有良好的效果，内毒素去除率超过90%；最终纯化所得BNP原液内毒素含量降至0.34EU/0.5mg，去除率高于99.99%，蛋白纯度高于98%；结论]所设计的四步层析纯化分离方法在纯化重组人脑利钠肽的同时也能高效的去除蛋白溶液中内毒素，所得原液内毒素的含量远远低于国家药典对注射剂内毒素含量的最低限制，为重组脑利钠肽的工业化生产奠定了基础。

Research on the control of endotoxin in the production of the recombinant human sodium peptide

Objective]The removal of endotoxin was controlled by means of column chromatography in the production of the recombinant human sodium peptide.Methods]Affinity chromatography of Chelating Sepharose FF,anion exchange chromatography of Q Sepharose ,?reversed phase chromatography of Source 15 and cation ex-change chromatography of SP Sepharose FF were applied consquently.The endotoxin levels were measured by Limulus Amebocyte Lysate gel-clot assay. Protein concentration and purity were determined by HPLC. Results]The column chrom-atography could remove endotoxin in great quantiti-es.Up to 99.99% endotoxin of BNP was removed and to a concentration of 0.34EU/0.5mg.Eventually the purity of BNP was up to 98%.Conclusion]This process was effective to purify BNP and remove endotoxin simultaneously,and?laid the foundation for the industrial production of the BNP.