摘要
目的建立单克隆抗体药物消光系数测定方法,用于单克隆抗体药物的鉴别和紫外分光光度法的定量分析,并对其进行初步评价。方法使用6 mol/L盐酸胍对抗体进行变性处理,打开抗体折叠的高级结构,将其当作是色氨酸、酪氨酸和"S-S"键的三元体系,通过检测相同浓度下折叠和非折叠抗体在紫外280 nm波长处的吸光值,计算抗体的消光系数,并进行方法的精密度验证。结果两种单克隆抗体m Ab1和m Ab2在280 nm波长处的消光系数平均值分别为1.702和1.387 m L/(mg·cm);两位实验员检测的消光系数各自相对标准偏差分别为0.48%和0.87%,总相对标准偏差为0.69%,均小于1%,表明方法精密度良好。结论建立的方法操作简单,不依赖于抗体浓度,不破坏其二硫键结构,结果准确,可用于单克隆抗体药物消光系数的测定。
Objective To establish and evaluate a method for determination of extinction coefficient（ε） of monoclonal antibody（m Ab）, and use for the identification of m Ab and quantitative analysis by ultraviolet spectrophotometry.Methods The m Ab sample was denatured with 6 mol/L guanidine hydrochloride（Gdn HCl）to unfold the higher spatial structure, and served as the ternary system consisting of tryptophan, tyrosine and disulfide bond in cystine. The extinction coefficient was calculated by measuring the absorbance of the same concentration of folded and unfolded m Ab at UV 280 nm,and the method separately was verified for precision. Results The mean extinction coefficients of m Ab1 and m Ab2 at280 nm were 1. 702 and 1. 387 m L/（mg·cm）, of which the RSDs by two personnel were 0. 48% and 0. 87%, respectively,while the total RSD was 0. 69%, indicating high precision of the method. Conclusion The method was simple and independent on the concentration of m Ab, of which the structure of the disulfide bond was undestroyed, and the result was accurate. The method may be used for determination of extinction coefficient of m Ab.
作者
孙开青
李文华
周冬梅
徐军
孙文正
杨彬
SUN Kai-qing;LI Wen-hua;ZHOU Dong-mei;XU Jun;SUN Wen-zheng;YANG Bin(Sunshine Lake Pharma Co. Ltd., Dongguan 523867, Guangdong Province, Chin)
出处
《中国生物制品学杂志》
CAS
CSCD
2018年第6期633-637,共5页
Chinese Journal of Biologicals
基金
广东省引进创新科研团队计划(201101Y0104990178)
关键词
单克隆抗体
消光系数
变性
朗伯比尔定律
Monoclonal antibody （mAb）
Extinction coefficient
Denaturation
Lambert-Beer law
