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目的建立中试制备重组人血清白蛋白-干扰素β(Recombinant human serum albumin-interferonβ,rHSA-IFNβ)融合蛋白的纯化工艺及质控方法。方法采用50 L发酵罐诱导表达rHSA-IFNβ融合蛋白,发酵液经超滤浓缩及脱盐后,再经BlueSepharose FF亲和层析、G25凝胶过滤层析和SP Sepharose FF阳离子交换层析纯化。SDS-PAGE(银染法)和HPLC测定融合蛋白纯度,Western blot分析反应原性,质谱法测定相对分子质量,Edman降解法测定N-末端氨基酸序列,等电聚焦电泳法测定等电点,细胞病变抑制法测定rHSA-IFNβ融合蛋白的生物学活性,其余检测项目均按《中国药典》三部(2010版)要求进行。结果纯化的3批融合蛋白纯度可达96%以上,具有人血清白蛋白和人干扰素β的双重反应原性,相对分子质量分别为89 070、89 035和88 669,N-末端氨基酸序列为:NH2-D-A-H-K-S,等电点为6.34,比活性分别为1.9×106、1.5×106和1.1×106IU/mg,其余各项指标均符合要求。结论已成功建立了中试制备rHSA-IFNβ融合蛋白的纯化工艺及质控方法。
Objective To establish a purification process and quality control method of recombinant human serum albumin-interferon β (rHSA-IFNβ) fusion protein by pilot scale. Methods The rHSA-IFNβ fusion protein was induced in a 50 L fermentor. The fermented broth was concentrated by ultrafiltration and desalted, and purified by Blue Sepharose FF affinity chromatography, G25 gel filtration chromatography and SP Sepharose FF cation exchange chromatography. The purity of the fusion protein was determined by SDS-PAGE (silver staining) and HPLC, the reaction was analyzed by Western blot, the molecular weight was determined by mass spectrometry, the N-terminal amino acid sequence was determined by Edman degradation method, the isoelectric point was determined by isoelectric focusing electrophoresis, The pathological inhibition method was used to determine the biological activity of rHSA-IFNβ fusion protein. The remaining test items were all conducted according to the requirements of “Chinese Pharmacopoeia” (2010 edition). Results The purity of the three batches of fusion proteins was over 96%, with the dual reactivity of human serum albumin and human interferon β. The relative molecular masses were 89 070, 89 035 and 88 669, respectively. The N-terminal amino acid sequence was : NH2-DAHKS, the isoelectric point was 6.34 and the specific activities were 1.9 × 106, 1.5 × 106 and 1.1 × 106IU / mg, respectively, and the rest of the indicators met the requirements. Conclusion The purification process and quality control method of rHSA-IFNβ fusion protein have been successfully established in pilot plant.