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| 单克隆抗体药物中游离巯基测定方法的建立 |
| Establishment of a method for determination of free sulfhydryl content in monoclonal antibody drugs |
| 投稿时间：2024-06-12 修订日期：2024-11-23 |
| DOI： |
| 中文关键词: 单克隆抗体药物 Ellman法 游离巯基含量 96孔酶标板 质量控制 |
| 英文关键词: Monoclonal antibody drugs Ellman method free sulfhydryl content 96-well enzyme labelling plate Quality control |
| 基金项目:国家重点研发计划-国家质量基础设施体系-蛋白类生物产制品质量检测和计量溯源技术 |
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| 中文摘要: |
| 目的 建立并验证一种利用96孔酶标板检测单克隆抗体药物中游离巯基含量的方法,可以快速、稳定、简便的测定单克隆抗体药物中的游离巯基含量。方法 利用Ellman法建立单克隆抗体药物中游离巯基的检测方法,并对该方法的专属性、线性、中间精密度、准确度和耐用性进行验证。结果 该方法专属性强,加入碘乙酰胺前后,吸光度值变化明显。在 0 ~ 45.5nmol.L-1范围内,游离巯基检测值和实测值线性相关,决定系数R2 > 0.99。两名实验人员分别在3个工作日对三批供试品的各6次独立测定的浓度的变异系数分别为1.33%、1.97%和2.42%,符合中间精密度的可接受标准(≤ 10%)。三批供试品的3个水平的加标溶液(10μmol.L-1,20μmol.L-1和40μmol.L-1)的平均回收率均在97.59% ~ 107.50%之间,且三批样品分别6次独立测定各水平浓度的变异系数均小于15%,符合准确度的验证要求(80%-120%)。不同仪器检测的回收率为97.46%。加入Ellman试剂15min和3h后分别进行检测,3h后游离巯基含量的回收率为97.75%。符合耐用性的可接受标准(90% ~ 110%)。结论 该方法各项指标均符合要求,可对单克隆抗体药物中游离巯基进行质量控制。 |
| 英文摘要: |
| Objective To establish and validate a method for the detection of free sulfhydryl content in monoclonal antibody drugs using a 96-well enzyme labeled plate, which can determine the free sulfhydryl content in monoclonal antibody drugs in a fast, stable and easy way. Methods The detection method for free sulfhydryl content in monoclonal antibody drugs was established by Ellman method, and the specificity, linearity, intermediate precision, accuracy and robustness of the method were validated. Results The specificity of the method was proved to be good, the absorbance changes obviously before and after adding iodoacetamide. The theoretical and measured free sulfhydryl content of the samples were linearly correlated in the range of 0 ~ 45.5 μmol.L-1, with a coefficient of determination R2 > 0.99. The CV of the 6 independent tests of the three batches samples by two staffs over three days were 1.97%, 1.33% and 2.42% respectively, which meets the acceptable criteria of intermediate precision(≤ 10%). The average recoveries of the three levels of spiked solutions (10μmol.L-1, 20μmol.L-1 and 40μmol.L-1) from the three batches of stock solutions were all in the range of 97.59% to 107.50%, and the CV of the three batches of stock solutions for each level of the spiked solutions were less than 15% for six independent measurements, which meets the acceptable criteria of accuracy (80%-120%). The recovery was 97.46% for different instruments. The assay was carried out 15 min and 3 h after the addition of Ellman reagent, the recovery of free sulfhydryl content after 3 h was 97.75%. which meets the acceptable criteria of robustness (90%-110%). Conclusion The method meets the requirements and can be used for quality control of free sulfhydryl content in monoclonal antibody drugs. |
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