rhEPOFc融合蛋白质量标准研究.docVIP

rhEPOFc融合蛋白质量标准研究 　　摘 要:目的:本研究采用的rhEPO-Fc融合蛋白是首次将编码人IgG Fc段的基因与编码人EPO的基因片段连接,并将其克隆至高表达质粒载体中,用该质粒转染CHO细胞进行分泌表达所得。通过对rhEPO-Fc融合蛋白的质量检验和实验方法参数的选择,建立rhEPO-Fc融合蛋白的质量标准。方法:采用细胞MTT比色法及Human EPO ELISA试剂盒测定rhEPO-Fc的体外生物学活性,Lowry法测定蛋白含量,SDS-聚丙烯酰胺凝胶电泳法测定相对分子质量,高效液相色谱法(HPLC)测定纯度,紫外分光光度法测定紫外吸收光谱,地高辛(DIG)标记的核酸探针法测定外源性DNA残留量,等电聚焦电泳法测定等电点,间苯二酚显色法测定唾液酸(SA)含量。结果:rhEPO-Fc的比活性为1.1×105IU/mg蛋白,蛋白含量为1.58mg/ml,相对分子质量约为59000道尔顿,纯度超过99.0%,紫外最大吸收峰约在280nm波长处,外源性DNA残留量小于10pg/mg,等电点为4.5～6.5,唾液酸含量为11.92mol/mol蛋白质。结论:所建立的质控方法已用于rhEPO-Fc制品的检定,可以有效地控制rhEPO-Fc制品的质量。 　　关键词:rhEPO-Fc 免疫球蛋白Fc段 融合蛋白 质量标准 　　中图分类号:R593 文献标识码:A 文章编号:1672-3791(2011)07(a)-0121-03 　　 　　Quality standards of rhEPO-Fc 　　 　　SHANG A-li,TANG Liang, CAO CHUAN-ping, et al (Shanghai Medipharm Biotech Co.Ltd,Shanghai 201203,China) 　　 　　Abstract:Objective:To establish the quality control standards for rhEPO-Fc based on optimized testing methods.Methods:The bioactivity of rhEPO-Fc was analyzed by MTT colorimetric assay and Human EPO ELISA kit,the Lowry method was used to measure the protein concentration,the reduced SDSwas used to determine the relative molecular weight,the protein purity was analyzed by HPLC and the non-reduced SDS,the ultraviolet absorption spectrum was recorded by ultraviolet spectroscopy,the high sensitivity hybridization assay for quantitation of residual DNA was carried out by using digoxin-labeled DNA,the isoelectric point (pI)was analyzed by IEF,quantitative estimation of sialic acid (SA)was detected by colorimetric resorcinol-hydrochloric method.Results:The specific activity,concentration,relative molecular weight and HPLC purity of rhEPO-Fc were 1.1×105IU/mg protein,1.58mg/ml, 59KD,and 99% respectively,its maximum ultraviolet absorption peak appeared at 280nm,the quantitation of residual DNA was less than10pg/mg,pI was 4.5-6.5,the detected sialic acid was 11.92mol/mol protein.Conclusion:The developed standards were suitable for the quality control of rhEPO-Fc product. 　　Key words:rhEPO-Fc;Fc fragment o