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

To Analyze the Differences of Biological Activity of Erythropoietin from Different Companies by Protein Stability Analysis and Micro-Scale Thermophoresis Technique

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

Key words:  erythropoietin  thermal stability  micro-scale thermophoresis (MST) technique  receptor affinity  biologic activity

摘要:

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、体外活性实验结果，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人促红素样品的热变性温度（Tm）、粒径、多分散系数（PDI）及变化情况；利用微量热泳动（MST）技术测定各企业人促红素与其受体作用时的解离平衡常数（KD）；利用Pearson检验对八家企业数据进行相关性分析。结果 八家企业人促红素样品Tm在54.84~59.64 ℃，粒径在3.71~4.93 nm，PDI在0.15~0.19，KD在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亲和性较差；D企业样品热稳定性最好，与EPOR亲和性最好。结论 各企业人促红素原液的分子间亲和力与其热稳定性呈正相关（P＜0.05）。结合相应制剂体内外生物活性结果，发现人促红素的分子亲和力和生物活性高度相关（P＜0.05）。蛋白质稳定性分析系统和MST技术可用于人促红素药物的活性分析，为蛋白类药物生物活性评价提供新的思路。

Abstract:

ABSTRACT：OBJECTIVE To investigate the thermal stability and receptor affinity of Erythropoietin（EPO） stock solutions from different companies. To evaluate the biological activities of formulations from different manufacturers combining the results of in vivo and in vitro experiments. METHODS The Protein Stability Analysis System was used to determine the melting temperature (Tm), particle size, and polymer dispersity index (PDI) of EPO samples during the heating process 25-95 ℃. Micro-Scale Thermophoresis (MST) was adopted to measure the dissociation constant (KD) of EPO samples with its receptors. Pearson test was used for correlation analysis of the data of eight enterprises. RESULTS The Tm of EPO samples from the eight companies were 54.84-59.64 ℃. The mean particle sizes were 3.71-4.93 nm. PDI were 0.15-0.19, and KD were 41.2-301.9 nmol·L-1. The particle size of the samples showed no dramatical change during the heating process. Samples from enterprise F and H had poor thermal stability and poor affinity with its receptors. Samples from enterprise D had the best thermal stability and the best affinity with its receptors. CONCLUSIONS The molecular affinity of EPO samples from various enterprises had a positive correlation with its thermal stability (P < 0.05). The molecular affinity of EPO had a high correlation with biological activity in vitro and in vivo (P < 0.05) combined biological activity in vitro and in vivo. The protein stability analysis system and MST technology can be used for the activity analysis of EPO. The method provide valuable references for the biological activity evaluation of protein drugs.