基于ASTM F2131-02标准试验的重组人骨形态发生蛋白-2体外生物活性检测方法的优化及验证  

Optimization and verification of a method for in vitro detection of bioactivity of recombinant human bone morphogenetic protein-2 based on ASTM F2131-02 standard test

作　　者：谢倩 甘琪 俞远满 钱江潮[1] XIE Qian;GAN Qi;YU Yuanman;QIAN Jiangchao(State Key Laboratory of Bioreactor Engineering,School of Bioengineering,East China University of Science and Technology,Shanghai 200237,China;不详)

机构地区：[1]华东理工大学生物工程学院生物反应器国家重点实验室,上海200237 [2]华东理工大学材料科学与工程学院教育部医用生物材料工程研究中心,上海200237

出　　处：《中国生物制品学杂志》2024年第9期1115-1121,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金(32071337)。

摘　　要：目的优化基于ASTM F2131-02标准试验的重组人骨形态发生蛋白-2(recombinant human bone morphogenetic protein-2,rhBMP-2)体外生物活性的检测方法,并进行验证。方法在美国试验材料学会(American Society for Testing Material,ASTM)建立方法的基础上,对检测细胞株(W-20-17及C2C12细胞)、rhBMP-2稀释方式(2及3倍系列稀释)、细胞培养时间(24、36、48、72 h)、细胞冷冻温度(-20及-80℃)进行优化,并验证优化方法的特异性、中间精密性、线性及耐用性。应用优化方法检测不同厂家来源rhBMP-2的比活性。结果确定采用C2C12细胞作为检测细胞;最佳rhBMP-2稀释方式为2倍系列稀释;最佳细胞培养时间为48 h;最佳细胞冷冻温度为-80℃。rhBMP-2样品中加入杂蛋白(牛血清白蛋白)时,C2C12细胞可特异性响应其中的rhBMP-2诱导作用;2名实验员于2个时间点共12次检测结果的CV为11.7%;rhBMP-2样品的理论效价在70%~142%范围内与实测效价呈良好的线性关系,拟合直线回归方程为:y=0.9831 x-0.0103,R^(2)为0.9964;采用4、11、21代次C2C12细胞检测rhBMP-2比活性的CV为5.2%。采用优化方法重复2次测定不同厂家来源rhBMP-2样品比活性的CV为1.3%~7.5%。结论优化的方法具有良好的特异性、中间精密性、线性及耐用性,可用于rhBMP-2体外生物活性的检测,为其研究、生产和应用提供了可靠的分析方法。Objective To optimize and verify an in vitro detection method for bioactivity of recombinant human bone morphogenetic protein-2(rhBMP-2)based on ASTM F2131-02 standard test.Methods Based on the method established by the American Society for Testing Material(ASTM),the detection cell lines(W-20-17 and C2C12 cells),dilution mode of rhBMP-2(2 and 3 times serial dilution),culture time(24,36,48 and 72 h),and freezing temperature(-20 and-80℃)were optimized,and the specificity,intermediate precision,linearity and durability of the optimized method were verified.The specific activity of rhBMP-2 from different manufacturers was detected using the optimized method.Results C2C12 cells were determined as detection cells;The best dilution method of rhBMP-2 was 2 times series dilution;The optimum cell culture timewas 48handtheoptimum freezingtemperatureofcellswas-80℃.C2C12 cells responded specifically to rhBMP-2 induction when adding heteroprotein(bovine serum albumin)to rhBMP-2;The CV of 12 detections by 2 experimenters at 2 time points was11.7%.The theoretical titer of rhBMP-2 showed a good linear relationship with the measured titer in the range of 70%-142%,and the fitting linear regression equation was:y=0.9831 x-0.0103,R^(2)=0.9964;The CV of the detected specific of rhBMP-2 using 4,11 and 21 passages of C2C12 cells was 5.2%.The CV of specific activity of rhBMP-2 samples from different manufacturers detected by the optimized method repeatedly for two times was 1.3%-7.5%.Conclusion The optimized method showed good specificity,intermediate precision,linearity and durability,and might be used to detect the bioactivity of rhBMP-2 in vitro,which provided a reliable analytical method for its research,production and application.

关 键 词：重组人骨形态发生蛋白-2 生物活性 比活性 

分 类 号：Q503[生物学—生物化学]