1.1本试验方法描述了用于测定 体外试验 使用小鼠基质细胞系W-20克隆17（W-20-17）的rhBMP-2的生物活性。该克隆来自W+小鼠株的骨髓基质细胞。 2. 1.2本试验方法（化验）已根据国际协调委员会化验验证指南进行鉴定和验证 3. （实验室间精密度除外）用于评估rhBMP-2的生物活性。这一点的相关性 体外 试验方法 体内 骨形成也进行了研究。在W-20生物测定中测得的反应，碱性磷酸酶诱导，已与rhBMP-2的异位成骨能力相关 体内 使用测试（UT）。通过两种甲硫氨酸的靶向过氧乙酸氧化部分或完全失活的rhBMP-2被用作比较活性的工具。通过肽图谱和质谱分析表明，用过氧乙酸氧化rhBMP-2对甲硫氨酸具有特异性靶向性。这些甲硫氨酸位于rhBMP上的疏水受体结合囊中- 2、将氧化样品与培养对照品和天然对照品进行比较。62、87、98和100 % 氧化样品的W-20活性水平为62、20、7和5 %, 分别地孵化和自然对照样品保持100 % 活动在UT中对样品进行了评估，并显示出类似的失活对骨形成活性的影响。62个样本 % 和20 % 与培养对照组相比，W-20测定中的活性表明骨形成水平降低，与W-20比活性的降低水平相似。 7和5例中很少或没有异位骨形成 % 活性rhBMP-2植入物。 1.3因此，受体结合位点中rhBMP-2分子的修饰降低了W-20和UT分析中的活性。这些数据表明，rhBMP-2上的单个受体结合域负责这两个过程 体外试验 和 体内 W-20生物测定是rhBMP-2成骨活性的相关预测因子。 1.4以国际单位制表示的数值应视为标准值。本标准不包括其他计量单位。 1.5 本标准并非旨在解决与其使用相关的所有安全问题（如有）。 本标准的用户有责任在使用前制定适当的安全和健康实践，并确定监管限制的适用性。 ====意义和用途====== 4.1尽管本试验方法可用于评估rhBMP-2粗制剂的生物活性，但其仅经验证可用于高纯度（>98 % 重组人骨形态发生蛋白-2的制备。

1.1 This test method describes the method used and the calculation of results for the determination of the in-vitro biological activity of rhBMP-2 using the mouse stromal cell line W-20 clone 17 (W-20-17). This clone was derived from bone marrow stromal cells of the W++ mouse strain. 2 1.2 This test method (assay) has been qualified and validated based upon the International Committee on Harmonization assay validation guidelines 3 (with the exception of interlaboratory precision) for the assessment of the biological activity of rhBMP-2. The relevance of this in vitro test method to in vivo bone formation has also been studied. The measured response in the W-20 bioassay, alkaline phosphatase induction, has been correlated with the ectopic bone-forming capacity of rhBMP-2 in the in vivo Use Test (UT). rhBMP-2 that was partially or fully inactivated by targeted peracetic acid oxidation of the two methionines was used as a tool to compare the activities. Oxidation of rhBMP-2 with peracetic acid was shown to be specifically targeted to the methionines by peptide mapping and mass spectrometry. These methionines reside in a hydrophobic receptor binding pocket on rhBMP-2. Oxidized samples were compared alongside an incubation control and a native control. The 62, 87, 98, and 100 % oxidized samples had W-20 activity levels of 62, 20, 7, and 5 %, respectively. The incubation and native control samples maintained 100 % activity. Samples were evaluated in the UT and showed a similar effect of inactivation on bone-forming activity. The samples with 62 % and 20 % activity in the W-20 assay demonstrated reduced levels of bone formation, similar in level with the reduction in W-20 specific activity, relative to the incubation control. Little or no ectopic bone was formed in the 7 and 5 % active rhBMP-2 implants. 1.3 Thus, modifications to the rhBMP-2 molecule in the receptor binding site decrease the activity in both the W-20 and UT assays. These data suggest that a single receptor binding domain on rhBMP-2 is responsible for both in-vitro and in-vivo activity and that the W-20 bioassay is a relevant predictor of the bone-forming activity of rhBMP-2. 1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. ====== Significance And Use ====== 4.1 Although the test method can be used for assessment of the bioactivity of crude preparations of rhBMP-2, it has only been validated for use with highly pure (>98 % by weight protein purity) preparations of rhBMP-2.