目的 本实验通过3种实验方法对1种单抗的消光系数进行实验确证并与理论消光系数进行比较研究。方法 根据氨基酸一级序列,计算出单抗在280 nm处的理论摩尔消光系数,通过质谱确定单抗相对分子质量,计算出理论消光系数;分别用3种方法测定单抗的蛋白质浓度,根据朗伯-比尔定律c=A/EL计算实验消光系数。方法一为利用尿素变性蛋白测定其非折叠状态(变性状态)的实验消光系数,计算得出折叠状态(天然状态)的消光系数;方法二通过酸水解蛋白,结合AccQ Fluor试剂盒利用HPLC对蛋白进行准确定量,计算该抗体的实验消光系数;方法三通过酸水解蛋白,利用氨基酸序列分析仪对蛋白进行准确定量,计算蛋白的实验消光系数。结果 理论消光系数E280为1.453 mL·mg-1·cm-1;3种实验方法确定的单抗消光系数分别为(1.442±0.009)、(1.529±0.032)、(1.458±0.019)mL·mg-1·cm-1,均值为(1.477±0.044)mL·mg-1·cm-1,RSD=2.979%(n=9)。研究结果显示,该单抗的理论消光系数与实验消光系数的差异较小(0.069%~7.708%)。结论 本实验运用3种方法确证单抗的实验消光系数,所建立的消光系数测定和验证方法可用于抗体药物蛋白含量的测定。
Abstract
OBJECTIVE To validate the experimental extinction coefficient of a monoclonal antibody with three METHODS and compare them with the theoretical value. METHODS The theoretical molar extinction coefficient was determined at 280 nm based on the primary sequence of the monoclonal antibody, and the theoretical extinction coefficient was calculated in combination with the molecular weight of the monoclonal antibody measured by mass spectrometry. The protein concentration of the monoclonal antibody was measured with three METHODS, and the experimental extinction coefficient was calculated according to Lambert-Beer's Law, ie, c=A/EL. Method one was calculating the extinction coefficient of the protein in its folded state (native state) based on the experimental extinction coefficient of the urea-denatured protein in its unfolded state (denatured state); method two was HPLC quantitation in combination with AccQ Fluor kit after acid hydrolysis of protein; method three was amino acid sequence analyzer quantitation after acid hydrolysis of the protein. RESULTS The theoretical extinction coefficient was 1.453 mL·mg-1·cm-1, and the experimental ones by the three METHODS were (1.442±0.009), (1.529±0.032), and (1.458±0.019) mL·mg-1·cm-1, respectively, with the average of (1.477±0.044) mL·mg-1·cm-1 and RSD of 2.979% (n=9), which demonstrated the small differences between the theoretical and experimental extinction coefficients (0.069%-7.708%). CONCLUSION The established determination and verification METHODS can be used to quantify the protein contents of monoclonal antibody products.
关键词
单克隆抗体 /
蛋白质含量 /
消光系数
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Key words
monoclonal antibody /
protein content /
extinction coefficient
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中图分类号：
R917
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参考文献
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脚注
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基金
国家“重大新药创制”科技重大专项资助项目(2014ZX09304311-001); 中国食品药品检定研究院学科带头人培养基金(2015X6)
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