重组人红细胞生成素大规模制备中反相色谱的条件优化

目的优化重组人红细胞生成素(rhEPO)大规模制备纯化中主要影响回收率的反相色谱的条件,提高回收率。方法采用反相纯化作为三步法纯化的第一步,利用R1型反相填料采用不同分步洗脱条件进行洗脱。结果优化后经第一步反相色谱所得rhEPO蛋白纯度达70%,再通过阴离子交换和凝胶过滤色谱纯化,rhEPO终产物的纯度可达100%,体内比活达1.91×105IU/mg,rhEPO蛋白的回收率提高了1.2倍。结论优化的反相纯化条件(30%乙醇-1.5 mol/L尿素,60%乙醇-3.0 mol/L尿素)能更有效的从培养上清中大量纯化出rhEPO,回收率高。获得的产品纯度高,体内外活性好。适合大规模纯化。

Optimized pilot purification process of reversed phase chromatography of recombinant human erythropoietin(rhEPO)

Purpose To optimize the pilot purification process of the recombinant human erythropoietin(rhEPO).Methods Culture supernatant was collected and purified by a three-step purification process-reversed phase,ion exchange and molecular sieve chromatography.Two stepwise elution conditions of reversed-phase chromatography in rhEPO were applied,and the reversed-phase chromatography is used as the first step of purification.Results The purity of sample was at least 70% by reversed-phase chromatography,but the sample purity was 100% and the sample biological activity was 1.91×105 IU/mg in vivo by later ion exchange and molecular sieve chromatography.Conclusion These results indicate that optimized purification process is rapid,and efficient.It will be more useful in large-scale production of rhEPO.