(1)背景:目前,在中国批准的用于注射产品的A型肉毒杆菌毒素在国内测试的唯一效力测定法是小鼠生物测定法(MBA)。中国神经毒素产品市场迅速扩大,但是MBA由于小鼠的个体差异而具有很高的变异性,以及注射部位的变化,除了有限的批次测试一个MBA。与MLD50法相比,由AbbVie开发的基于细胞的效价测定法(CBPA)用于检测单纯碱毒素A(BOTOX)的效价,不仅不使用任何实验动物,而且可以节省大量时间和成本.由于在中国用CBPA替代mLD50测定所带来的巨大好处,CBPA方法已经转移,已验证,并进行了交叉验证,以证明两种效力方法的等效性。(2)方法:分化的SiMa细胞用BOTOX样品和参考标准处理,并且使用Chemi-ECLELISA定量细胞裂解物中裂解的SNAP25197。4-PL模型用于数据拟合和样品相对效力计算。方法的准确性,线性度重复性,和中间精度在标记权利要求的50%至200%范围内确定。两种效力方法(CBPA和mLD50)的统计等效性最初是通过将AbbVieCBPA数据与NIFDCmLD50数据在总共167个商业BOTOX批次(85个50U批次和82个100U批次)上进行比较来证明的。此外,通过这两种方法的等效性,重新测试了6批onabotulinumtoxinA(3批50U和3批100U)作为交叉验证.(3)结果:总体测定的准确性和中间精密度分别为104%和9.2%,和斜坡,R-正方形,线性和Y截距分别为1.071、0.998和0.036。重复性确定为6.9%。符合可接受精度标准的范围,线性度精确度被证明是标记索赔的50%到200%。使用边距的95%等效统计检验[80%,125%]表明CBPA和mLD50方法对于两种BOTOX强度是等效的(即,50U和100U)。来自交叉验证的相对效力数据在≥80%至≤120%的范围内。(4)结论:CBPA符合所有验收标准,相当于mLD50。用CBPA替换mLD50在确保安全性和有效性方面是合理的,以及动物的利益。
(1) Background: At present, the only potency assay approved in
China for the in-country testing of botulinum toxin type A for injection products is the mouse bioassay (MBA). The Chinese market for neurotoxin products is rapidly expanding, but MBAs are subject to high variability due to individual variations in mice, as well as variations in injection sites, in addition to the limited number of batches tested for one MBA. Compared with the mLD50 method, the cell-based potency assay (CBPA) developed for the potency testing of onabotulinumtoxinA (BOTOX) by AbbVie not only does not use any experimental animals but also allows for significant time and cost savings. Due to the significant benefits conferred by the replacement of the mLD50 assay with CBPA in
China, the CBPA method has been transferred, validated, and cross-validated to demonstrate the equivalence of the two potency methods. (2) Methods: The differentiated SiMa cells were treated with both BOTOX samples and the reference standard, and the cleaved SNAP25197 in the cell lysates was quantified using Chemi-ECL ELISA. A 4-PL model was used for the data fit and sample relative potency calculation. The method accuracy, linearity, repeatability, and intermediate precision were determined within the range of 50% to 200% of the labeled claim. A statistical equivalence of the two potency methods (CBPA and mLD50) was initially demonstrated by comparing the AbbVie CBPA data with NIFDC mLD50 data on a total of 167 commercial BOTOX lots (85 50U lots and 82 100U lots). In addition, six lots of onabotulinumtoxinA (three 50U and three 100U) were re-tested as cross-validation by these two methods for equivalence. (3) Results: The overall assay\'s accuracy and intermediate precision were determined as 104% and 9.2%, and the slope, R-square, and Y-intercept for linearity were determined as 1.071, 0.998, and 0.036, respectively. The repeatability was determined as 6.9%. The range with the acceptable criteria of accuracy, linearity, and precision was demonstrated as 50% to 200% of the labeled claim. The 95% equivalence statistic test using margins [80%, 125%] indicates that CBPA and mLD50 methods are equivalent for both BOTOX strengths (i.e., 50U and 100U). The relative potency data from cross-validation were within the range of ≥80% to ≤120%. (4) Conclusions: The CBPA meets all acceptance criteria and is equivalent to mLD50. The replacement of mLD50 with CBPA is well justified in terms of ensuring safety and efficacy, as well as for animal benefits.