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
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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、72h)、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=0.983 1 x-0.010 3,R2为0.996 4;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 mor-phogenetic 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 time was 48 h and the optimum freezing temperature of cells was-80℃.C2C12 cells responded specifically to rhBM P-2 induction when adding heteroprotein(bovine serum albumin)to rhBMP-2;The CV of 12 detections by 2 experimenters at 2 time points was 11.7％.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=0.983 1 x-0.010 3,R2=0.996 4;The CV of the detected specific ofrhBMP-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 opti-mized 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.
Recombinant human bone morphogenetic protein-2(rhBMP-2)BioactivitySpecific activity
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