目的 建立一种适用于阿达木单抗参照药和生物类似药血药浓度检测的单一生物分析方法。方法 以人肿瘤坏死因子α(hTNF-α)作为捕获抗原,以羊抗人IgG作为检测抗体建立间接酶联免疫分析法,从标准曲线与定量范围、精密度与准确度、钩状效应、选择性以及稳定性方面进行方法学系统验证,参照行业白皮书推荐的均衡实验设计进行参照药和生物类似药的方法一致性评估。结果 两种生物类似药与参照药的类似性比较显示标准曲线重合,无统计学差异,生物类似药与参照药质控样本的回算偏差的差异值小于5%,90%置信区间小于15%;方法学系统验证显示生物类似药和参照药的批间精密度小于20%,批间准确度均小于10%,总误差符合要求;方法在1~1 000 μg·mL-1内无钩状效应,不同个体样本选择性验证满足要求,在室温放置24 h,4 ℃放置5 d,冻融3次以及-80 ℃冻存1个月的条件下样本均稳定。结论 本研究建立了一种阿达木单抗血药浓度检测的单一生物分析方法,并通过参照药和生物类似药分析方法相似性验证,可采用该单一生物分析方法对生物类似药进行治疗药物监测。
Abstract
OBJECTIVE To develop a single bioanalysis method of adalimumab reference and two biosimilars in human plasma and carry out similarity assessment. METHODS Human tumor necrosis factor-α(hTNF-α)was used as capture antigen, AND goat anti-human IgG-HRP was used as detection antibody to establish indirect enzyme-linked immunosorbent assay method. The validation wasperformed from the aspects of standard curve and quantitative range, accuracy and precision, hook effect, selectivity, and stability. According to the balanced designed experiments recommended by the white paper, the method consistency of reference and its biosimilars was evaluated. RESULTS The comparison of the similarity between adalimumab reference and its biosimilars showed that the standard curves basically overlapped, and there was no statistic difference. The difference value of the back-calculated deviation between reference and its biosimilars quality control samples was less than 5% and the 90% confidence interval for the difference was less than 15%. The methodology systematic validation showed that the interbatch precision of reference and its biosimilars was less than 20%, and the interbatch accuracy was less than 10%, and the total error met the requirements. There was no hook effect in the assay range of 1-1 000 μg·mL-1. The selectivity of different individual samples met the requirements. The samples were stable at room temperature for 24 hours, 4 ℃ for 5 days, freeze-thaw for 3 times and -80 ℃ for 1 month. CONCLUSION In this study, a single bioanalysis method of adalimumab in human plasma is established. Through the verification of the similarity between reference and two biosimilars, the single bioanalysis method can be used for therapeutic drug monitoring.
关键词
阿达木单抗 /
酶联免疫吸附分析法 /
生物类似药 /
方法相似性验证
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Key words
adalimumab /
enzyme-linked immunosorbent assay /
biosimilar /
methodology similarity validation
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中图分类号：
R969
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参考文献
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脚注
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基金
国家自然科学基金青年项目资助(82003857);苏州市姑苏卫生人才计划项目资助(GSWS2019001);苏州市科技发展计划项目资助(SYSD2019176)
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