离子交换层析结合分子排阻色谱法测定注射用尿激酶中分子组分比  

Determination of molecular fraction ratio in urokinase for injection by ion exchange chromatography combined with size exclusion chromatography

作　　者：严翠霞 尹红锐 史芳亮 陈钢 郑璐侠 邵泓 YAN Cui-xia;YIN Hong-rui;SHI Fang-liang;CHEN Gang;ZHENG Lu-xia;SHAO Hong(Shanghai Institute for Food and Drug Control,Shanghai 201203,China;NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies,Shanghai 201203,China)

机构地区：[1]上海市食品药品检验研究院,上海201203 [2]国家药监局治疗类单抗质量控制重点实验室,上海201203

出　　处：《药物分析杂志》2023年第1期1-11,共11页Chinese Journal of Pharmaceutical Analysis

基　　金：国家药典委员会课题(2018H081)。

摘　　要：目的:建立测定注射用尿激酶分子组分比的离子交换层析样品前处理方法结合分子排阻色谱法的分析方法,以解决长期以来制剂标准中无法控制分子组分比的难题,并对国内6家企业的产品质量进行了考察。方法:根据尿激酶与人血白蛋白等电点的差异,通过离子交换层析去除样品中人血白蛋白辅料的干扰,采用分子排阻色谱法测定。采用TSKgel G2000SWXL色谱柱(300 mm×7.8 mm, 5μm),流动相为0.1 mol·L^(-1)磷酸二氢钠缓冲液(pH 3.0),流速为0.5 mL·min^(-1),柱温为35℃,检测波长为280 nm,进样量为50μL。结果:尿激酶质量浓度在0.05~5.1 mg·mL^(-1)范围内,高分子量尿激酶与低分子量尿激酶线性关系均良好(r≥0.998 0);检测限分别为1.5μg·mL^(-1)与5.1μg·mL^(-1),定量限分别为5.1μg·mL^(-1)与16.9μg·mL^(-1);精密度、重复性、24 h稳定性试验的RSD均<4.0%;方法的回收率为92.5%~97.0%;18批样品中高分子量尿激酶与低分子量尿激酶的相对含量范围分别为82.8%~96.5%与3.5%~17.2%。结论:建立的离子交换层析结合分子排阻色谱法可用于注射用尿激酶中分子组分比的测定,弥补了注射用尿激酶现行国家标准中分子组分比测定的空白。Objective:To establish a determination method for molecular fraction ratio in the sample pretreatment of urokinase for injection using ion exchange chromatography(IEC) combined with size exclusion chromatography(SEC) so as to find a solution to fix the ratio of molecular fraction could not be controlled in the preparation’s quality standard for a long time, the product quality of six domestic companies was investigated. Methods: Human serum albumin was removed by IEC for its distinguishing isoelectric point, and then the urokinase in the rest samplewere measured by SEC. The chromatographic column was TSKgel G2000SWXL column(300 mm×7.8 mm, 5 μm) with 0.1 mol·L^(-1)sodium dihydrogen phosphate buffer(pH 3.0) as mobile phase, the flow rate was 0.5 mL·min^(-1), the column temperature was 35 ℃. The detection wavelength was 280 nm and the injection volume was 50 μL. Results: The urokinase at high and low molecular mass showed good linear relationship(r≥0.998 0) when the concentration of urokinase was in the range of 0.05 to 5.1 mg·mL^(-1). The detection limit of high molecular mass urokinase and low molecular mass urokinase were 1.5 μg·mL^(-1)and 5.1 μg·mL^(-1), respectively, and the limit of quantification were 5.1 μg·mL^(-1)and 16.9 μg·mL^(-1),respectively.The RSDs of precision, repeatability, stability test in 24 h were less than 4.0%. The recovery was 92.5%-97.0%, the relative content range of high molecular mass urokinase in all 18 batches of samples was 82.8%-96.5%, and low molecular mass urokinase was 3.5%^(-1)7.2%. Conclusion: The established ion exchange chromatography combined with size exclusion chromatography is applicable for the determination of molecular fraction in the preparation of urokinase for injection, and fills the gap in the current quality standards of urokinase for injection.

关 键 词：注射用尿激酶 高分子量尿激酶 低分子量尿激酶 分子组分比 离子交换层析 分子排阻色谱 

分 类 号：R917[医药卫生—药物分析学]