生色底物法测定尿激酶原料与制剂效价的可行性分析  

Feasibility Analysis of Chromophore Substrate Method for Assay of Urokinase API and Preparation

作　　者：孙珍 严翠霞 段徐华 郑璐侠 陈钢 邵泓 Sun Zhen;Yan Cuixia;Duan Xuhua;Zheng Luxia;Chen Gang;Shao Hong(Shanghai Institute for Food and Drug Control,SFDA Key Laboratory of Quality Control of Therapeutic Monoclonal Antibodies,Shanghai 201213,China)

机构地区：[1]上海市食品药品检验研究院国家药监局治疗类单抗质量控制重点实验室,上海201213

出　　处：《中国药师》2021年第3期566-570,共5页China Pharmacist

摘　　要：目的:建立生色底物法测定尿激酶和注射用尿激酶效价的新方法,对生色底物法的反应条件进行优化后,进行方法学验证,并在统计学意义上与现行的气泡上升法进行方法一致性评价。方法:对反应温度、pH、反应时间、底物浓度及酶反应浓度进行考察,确立0.3 mmol·L^(-1)L-焦谷氨酰甘氨酰-L-精氨酸-对硝基苯胺盐酸盐溶液作为底物,水浴温度(35±0.2)℃,准确反应30 min,加入40%醋酸溶液终止反应后,在405 nm波长处进行吸光度的测定。采用统计学软件对两种方法的测定结果进行Bland-Altman图分析。结果:尿激酶在6~50单位/L浓度范围内,吸光度呈良好线性关系(r=0.999 8),加样回收率在99.9%~101.5%之间,RSD为0.7%~1.5%(n=3),统计学分析结果表明与现行的气泡上升法测定结果具有一致性。结论:生色底物法操作简单方便,结果客观,重复性好,精密度高,生色底物法可以用作尿激酶与注射用尿激酶效价测定的现行气泡上升法的替代方法。Objective: To establish a new method for the titer determination of urokinase for urokinase API and preparation for injection,investigate the reaction conditions and methodology of chromogenic substrate method,and perform consistency evaluation statistically with the bubble rise method.Methods: The reaction temperature,pH value,reaction time,substrate concentration and enzyme concentration were investigated.0.3 mmol·L^(-1) L-Pyroglutamylglycyl-L-arginine-p-nitroaniline hydrochloride solution was used as a substrate,and the water bath temperature was(35±0.2) ℃ . The reaction was continuously for 30 min.After adding 40% acetic acid solution to stop the reaction,the absorbance was measured at 405 nm using a UV-visible spectrophotometer.MedCalc statistical softwave was used for the statistical analysis of Bland-Altman plots of the two methods.Results: Urokinase was within a concentration range of 6-50 units·ml^(-1),and the absorbance showed a good linear relationship(r= 0.999 8).The sample recoveries were 99.9%-101.5%,and the RSDs were 0.7%-1.5%(n = 3).The statistical analysis showed that the results were consistent with those of current bubble rise method.Conclusion: The chromogenic substrate method is simple and convenient to operate,which is objective and reproducible with high precision.The chromogenic substrate method can be used as a better alternative method for the titer determination of urokinase API and preparation for injection.

关 键 词：尿激酶 注射用尿激酶 效价测定 生色底物法 Bland-Altman分析 

分 类 号：R917[医药卫生—药物分析学]