均相时间分辨荧光法检测德谷胰岛素生物学活性  

Detection of biological activity of insulin degludec by homogeneous time-resolved fluorescence

作　　者：高婧 王绿音 李晶[2] 吕萍[2] 高向东[1] 梁成罡[2] GAO Jing;WANG Lü-yin;LI Jing;LÜPing;GAO Xiang-dong;LIANG Cheng-gang(College of Life Science and Technology,China Pharmaceutical University,Nanjing 211198,China;State Key Laboratory of Drug Regulatory Science,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,National Institutes for Food and Drug Control,Beijing 102629,China)

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京211198 [2]中国食品药品检定研究院,药品监管科学全国重点实验室,国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室,北京102629

出　　处：《药学学报》2024年第12期3347-3353,共7页Acta Pharmaceutica Sinica

基　　金：2024年度国家药品标准制修订研究课题(2024S09)

摘　　要：本研究以CHO_INSR_1284转基因细胞系为靶细胞,利用均相时间分辨荧光技术建立德谷胰岛素生物学活性检测方法。对关键参数进行优化,并依据中国药典(2020版四部通则9401、1431)进行验证。结果表明,德谷胰岛素在本方法中存在良好的剂量效应关系,符合四参数曲线。经优化,细胞种板密度为每毫升3.5×10^(5)个,德谷胰岛素初始浓度为57.18μg·m L^(-1),稀释倍数为4倍,刺激时间为45 min,孵育时间为4 h。本方法专属性强,5个效价水平几何变异系数GCV%为4.1%~10.6%,线性拟合的直线回归方程为y=1.015x-0.0277,R^(2)=0.9996,结果证明此方法具有的较好中间精密度和线性。且回归项非常显著(P<0.01),偏离平行项和模型失拟项均不显著(P≥0.05),均符合中国药典要求。本研究建立了一种基于均相时间分辨荧光技术的德谷胰岛素生物学活性检测方法,该方法可用于德谷胰岛素类产品的生物学活性评价和质量控制。In this study,the CHO_INSR_1284 transgenic cell line was employed as the target cell,utilizing homogeneous time-resolved fluorescence technology to establish a method for detecting the biological activity of insulin degludec.Key parameters were optimized,and validation was conducted in accordance with general principles 9401 and 1431 of the fourth section of the 2020 edition of the Chinese Pharmacopoeia.Results indicated a good dose-response relationship for insulin degludec in this method,aligning with a four-parameter curve.Following optimization,the cell seeding density was set at 3.5×10^(5) cell·m L^(-1),the initial concentration of insulin degludec at 57.18μg·m L^(-1),with a four-fold dilution,a stimulation period of 45 minutes,and an incubation duration of 4 hours.This method demonstrated strong specificity,with the geometric variation coefficient (GCV%) for the five potency levels ranging from 4.1%to 10.6%.The linear regression equation from the linear fitting was y=1.015x-0.027 7,and R^(2)=0.999 6.The results confirmed the method's good intermediate precision and linearity.The regression term was highly significant (P<0.01),neither the deviation from parallel terms nor the model mismatch terms were significant (P≥0.05),complying with general rule 1431 of the fourth section of the 2020edition of the Chinese Pharmacopoeia.This study established a method for detecting the biological activity of insulin degludec using homogeneous time-resolved fluorescence technology,suitable for evaluating the biological activity and quality control of insulin degludec products.

关 键 词：生物学活性 德谷胰岛素 脂肪酸修饰胰岛素 转基因细胞 均相时间分辨荧光技术 

分 类 号：R917[医药卫生—药物分析学]