离子色谱法测定注射用阿糖苷酶α糖原水解动力学关键参数  

Establishment of a method for determining the key parameters of hydrolysis kinetics of acid α-glucosidase for injection by ion chromatography

作　　者：胡馨月 孔嘉浩 孙悦[1] 王绿音 张孝明 吕萍[1] 梁成罡[1] 李晶[1] HU Xin-yue;KONG Jia-hao;SUN Yue;WANG Lü-yin;ZHANG Xiao-ming;LÜPing;LIANG Cheng-gang;LI Jing(National Institutes for Food and Drug Control,State Key Laboratory of Drug Regulatory Science,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs,Beijing 102629,China;Beijing Institute of Petrochemical Technology,Beijing 102627,China)

机构地区：[1]中国食品药品检定研究院,药品监管科学全国重点实验室,国家卫生健康委生物技术产品检定方法及其标准化重点实验室,国家药品监督管理局生物制品质量研究与评价重点实验室,国家药品监督管理局化学药品质量研究与评价重点实验室,北京102629 [2]北京石油化工学院,北京102627

出　　处：《药学学报》2024年第12期3361-3366,共6页Acta Pharmaceutica Sinica

基　　金：药品监管科学全国重点实验室第一批课题项目(2023SKLDRS0108);药品监管科学全国重点实验室第二批课题项目(2024SKLDRS0203);中国食品药品检定研究院化学药品检定所重点实验室项目(2024HYZX23)。

摘　　要：采用Dionex CaboPac^(TM) PA10 BioLC^(TM) Analyical 2 mm×250 mm色谱柱,配以保护柱(Dionex CaboPac^(TM )PA10 BioLC^(TM) Guard 2 mm×50 mm);以100 mmol·L^(-1)氢氧化钠溶液为淋洗液;流速为0.25 mL·min^(-1);样品盘温度:35℃。采用脉冲安培检测器,波形为:Gold CWE,Ag-AgCl RE,Carbo,Quad。以8个浓度的糖原底物(0.31、1.25、2.5、5、10、20、30和40 mg·mL^(-1))培养样品。在5个不同时间点(T0~T4)时测定葡萄糖浓度。T1~T4的葡萄糖浓度减去T0时的葡萄糖浓度,在不同糖原底物浓度下,计算反应速率。利用米氏方程,绘制这些反应速率与底物浓度的曲线。以V_(max) (nmol·mg^(-1)·min^(-1))和K_(m) (mg·mL^(-1))表示动力学参数。葡萄糖标准曲线(线性范围:1.25~500μmol·L^(-1)),葡萄糖标准曲线的系数R^(2) (n=6)的RSD为0.1%,标准曲线斜率RSD为2.2%;定量限平均值为0.14μmol·L^(-1),检测限平均值为0.05μmol·L^(-1);供试品重复3次单独实验K_(m)的RSD为4.4%,V_(max)的RSD为4.6%,方法耐用性良好。建立离子色谱法在线自动化测定注射用阿糖苷酶α糖原水解动力学的方法,该方法精密度、重复性和耐用性良好,可以用于注射用阿糖苷酶α糖原水解动力学的检测,并且对重组酶替代疗法酶类的糖原动力学评价具有参考价值。The Dionex CaboPac^(TM) PA10 BioLC^(TM) Analyical 2 mm×250 mm column was used with a protective column (Dionex CaboPac^(TM) PA10 BioLC^(TM) Guard 2 mm×50 mm).100 mmol·L^(-1) sodium hydroxide solution was used as eluent;the flow rate was 0.25 mL·min^(-1).Sample tray temperature:35℃.The pulse amperometric detector was adopted,and the waveform was Gold CWE,Ag-AgCl RE,Carbo,Quad.The samples were cultured with8 concentrations of glycogen substrates (0.31,1.25,2.5,5,10,20,30,and 40 mg·mL^(-1)).D-Glucose concentrations were measured at 5 different time points (T0,T1,T2,T3 and T4).The glucose concentration from T1 to T4 minus the glucose concentration at T0.The reaction rate was calculated at different glycogen substrate concentrations.These reaction rates are plotted against substrate concentrations using Michaelis-Menten equation.The kinetic parameters were expressed as V_(max) (nmol·mg^(-1)·min^(-1)) and K_(m) (mg·mL^(-1)).The RSD of glucose standard curve R^(2)(n=6,linear range:1.25-500μmol·L^(-1)) was 0.1%and the RSD (n=6) of the slope of the standard curve was2.2%.The mean limit of quantitation was 0.14μmol·L^(-1),and the mean limit of detection was 0.05μmol·L^(-1).The RSD of K_(m) and V_(max) were 4.4%and 4.6%respectively in three separate experiments.The durability of the method was good.The method was developed for the on-line automatic determination of the hydrolysis kinetics of acidα-glucosidase (GAA) for injection by ion chromatography.The method has good precision,repeatability and durability,and can be used for the determination of glycogen hydrolysis kinetics of GAA for injection,and could reference value for the enzyme kinetics evaluation of recombinant enzyme replacement therapy.

关 键 词：注射用阿糖苷酶α 离子色谱法 脉冲安培检测器 米氏方程 酶动力学 在线自动化 

分 类 号：R917[医药卫生—药物分析学]