基于蛋白质稳定性分析系统和微量热泳动技术分析不同企业人促红素的生物活性差异  

Differences of Biological Activity of Erythropoietin from Different Companies Based on Protein Stability Analysis and Micro-scale Thermophoresis Technique

作　　者：李佳奇 何睿 张一童 邬忠康 刘贝贝 刘海静 王嫦鹤[1,2,5] LI Jiaqi;HE Rui;ZHANG Yitong;WU Zhongkang;LIU Beibei;LIU Haijing;WANG Changhe(Shaanxi Institute for Food and Drug Control,Xi’an 710065,China;Shaanxi University of Traditional Chinese Medicine,Xianyang 712046,China;Department of Pharmacy,Honghui Hospital Affiliated to Xi'an Jiaotong University,Xi’an 710054,China;Nanotemper Technology Co.,Ltd.,Beijing 100027,China;Shaanxi Key Laboratory of Food and Drug Safety Monitoring,Xi’an 710065,China)

机构地区：[1]陕西省食品药品检验研究院,西安710065 [2]陕西中医药大学,陕西咸阳712046 [3]西安交通大学附属红会医院药剂科,西安710054 [4]诺坦普科技有限公司,北京100027 [5]陕西省食品药品安全监测重点实验室,西安710065

出　　处：《中国现代应用药学》2024年第18期2556-2562,共7页Chinese Journal of Modern Applied Pharmacy

基　　金：陕西省创新能力支撑计划(2020PT-041);陕西省社会发展科技攻关项目(2021SF-304)。

摘　　要：目的 探讨不同企业人促红素(erythropoietin,EPO)原液热稳定性及其受体亲和力的强弱,结合体内、体外活性试验结果,验证不同企业制剂的生物活性差异。方法 利用蛋白质稳定性分析系统测定在25~95℃升温过程中不同企业EPO样品的热变性温度(T_(m))、粒径、多分散系数(polymer dispersity index,PDI)及变化情况;利用微量热泳动(micro-scale thermophoresis,MST)技术测定各企业EPO与其受体作用时的解离平衡常数(K_(D));利用Pearson检验对8家企业数据进行相关性分析。结果 8家企业EPO样品T_(m)为54.84~59.64℃,粒径为3.71~4.93 nm,PDI为0.15~0.19,K_(D)为41.37~301.40 nmol·L^(-1),样品粒径在升温过程中均未出现剧烈变化。其中F和H企业样品热稳定性较差,与EPOR受体亲和性较差;D企业样品热稳定性最好,与EPOR受体亲和性最好。结论 各企业EPO原液的分子间亲和力与其热稳定性高度相关,也与其生物活性高度相关。MST技术可用于EPO药物的生物活性分析,结合蛋白质稳定性分析系统,为蛋白类药物的生物活性评价和稳定性评价提供新的思路。OBJECTIVE To investigate the thermal stability and receptor affinity of erythropoietin(EPO)stock solutions from different companies and evaluate their biological activities through both in vivo and in vitro experiments.METHODS The protein stability analysis system was employed to ascertain the melting temperature(T_(m)),particle size and polymer dispersity index(PDI)of EPO samples as the temperature increased from 25℃ to 95℃.Micro-scale thermophoresis(MST)was utilized to measure the affinity constant(K_(D))of EPO in relation to its receptors.Pearson correlation test was conducted to analyze the data from eight enterprises.RESULTS The T_(m) of EPO samples from the eight companies ranged from 54.84 to 59.64℃.The mean particle sizes were 3.71−4.93 nm.PDI were 0.15−0.19,and K_(D) were 41.37−301.40 nmol·L^(−1).The particle size of the samples exhibited no significant changes during the heating process.Samples from enterprise F and H had poor thermal stability and low affinity with its receptors.Samples from enterprise D displayed the best thermal stability and the best affinity with its receptors.CONCLUSION The molecular affinity of EPO samples from various enterprises has a positive correlation with their thermal stability and biological activity molecular affinity of EPO had high correlation with biological activity both in vitro and in vivo(P<0.05).MST technology is effective for evaluating the activity of EPO,combined with the protein stability analysis system,providing valuable reference for the bioactivity evaluation and stability evaluation of protein drugs.

关 键 词：人促红素 热稳定性 微量热泳动技术 亲和力 生物活性 

分 类 号：R945[医药卫生—微生物与生化药学]