MO7e细胞增殖法检测注射用重组人促血小板生成素模拟肽-Fc融合蛋白生物活性方法的建立和验证

Development and validation of MO7e cell proliferation assay method for detection of bioactivity of recombinant human thrombopoietin mimetic peptide-Fc fusion protein for injection

目的:建立并验证用于检测注射用重组人促血小板生成素模拟肽-Fc融合蛋白(recombinant human thrombopoietin mimetic peptide-Fc fusion protein,TMP-Fc)生物活性的MO7e细胞增殖法,用于TMP-Fc的质量控制。方法:以人巨核细胞白血病细胞株MO7e作为基质细胞,不同浓度的TMP-Fc与MO7e细胞作用,与其表面血小板生成素(thrombopoietin,TPO)受体结合,刺激细胞增殖,通过比色法测定细胞增殖情况来评价TMP-Fc的生物学活性,其结果用质控参考品进行校正。从显色方法、作用时间、TMP-Fc稀释率等多个方面对实验条件进行优化。对方法的专属性、线性与范围、准确度及精密度进行验证。结果:MO7e细胞对TMP-Fc有很好的剂量反应关系,方法专属性良好;线性范围为0.006 4～2 500 ng·m L-1,相关系数≥0.99;回收率在80%～120%之间。3批原研品和3批TMP-Fc经3次重复测定,其RSD均在15%之内,说明方法精密度良好;其活性分别为(193 152±6 799)～(219 187±11 390)U·mg-1和(207 822±30 423)～(228 549±15 865)U·mg-1,采用One-way ANOVA统计分析,组间P值大于0.05,说明TMP-Fc生物学活性与原研品相似。结论:建立并验证测定TMP-Fc生物活性的MO7e细胞增殖法,该方法具有较好的专属性、准确度和精密度,可用于TMP-Fc的生物学活性评价和质量控制。

Objective：To develop and validate an optimized MO7e cell proliferation assay method for detecting the bioactivity of TMP-Fc for injection and to control the quality of TMP-Fc.Methods：A human MO7e leukemic cell was used as the stromal cell.Different concentrations of TMP-Fc were combined with thrombopoietin（TPO） receptor to stimulate cell proliferation.The biological activity of TMP-Fc was evaluated by colorimetric assay.The results were calibrated with reference quality control.The experimental conditions were optimized from several factors including colorimetric method, interaction time and dilution factor of TMP-Fc.The developed method was validated for specificity, linear range, accuracy and precision. Results：MO7e cells had a good dose-response relationship to TMP-Fc, and the method had good specificity.The detection linear range was from 0.006 4 to 2 500 ng·mL^-1, the linear correlation coefficient was greater than 0.99, and the mean recoveries ranged between 80% and 120%.The results of three batches of launched products and three bathes of samples were（193 152±6 799） -（219 187±11 390） U·mg^-1 and（207 822±30 423） -（228 549±15 865） U·mg^-1, respectively, RSD ≤ 15%.Statistical analysis（One-way ANOVA） result was P value〉0.05.The results demonstrated that the assay had good precision and TMP-Fc bioactivity was consistent with original products.Conclusion：A MO7e cell proliferation assay method is successfully established following optimization, characterized by good specificity, accuracy and precision, and this method can be used for the bioactivity assessment and quality control for TMP-Fc.