罕见病酶替代疗法药物重组人α-半乳糖苷酶A酶活性测定方法研究

Activity Assay of Recombinant Humanα-Galactosidase A for Enzyme Replacement Therapy of Rare Diseases

目的基于酶反应动力学理论对重组人α-半乳糖苷酶A(recombinant humanα-galactosidase A,rhα-GAL)酶活性测定反应条件进行系统研究,建立更灵敏简便、准确稳定的rhα-GAL酶活性测定方法。方法优化反应体系后的最适条件为:以50 mmol·L^(-1)对硝基苯基-α-D-吡喃半乳糖苷的为底物,酶质量浓度为1.67μg·mL^(-1),于37℃水浴准确反应15 min后,加入甘氨酸缓冲液(pH10.5)终止反应,采用酶标仪在400 nm波长处测定产物对硝基苯酚的光密度值。结果该方法专属性良好,rhα-GAL在(0.83~2.51 mg·mL^(-1))(r=0.9998)质量浓度范围内与酶促反应速率呈良好的线性关系;rhα-GAL在50%、80%、100%、125%及150%浓度水平验证溶液的回收率在94.2%~101.8%范围内(n=18),测定结果的变异系数(CV)在2.0%~5.5%(n=18);同一份供试品溶液的12次独立测定结果的CV为2.21%;考察反应体系中的水浴温度、反应时间和底物浓度微小变化对测定结果的影响,结果表明方法的耐用性较好;复溶后的样品于2~8℃存放48 h稳定性较好;水解产物对硝基苯酚在(0.01~0.15 mmol·L^(-1))(r=0.9997)范围内与光密度呈良好的线性关系,5个浓度水平的回收率在94.9%~105.1%之间(n=9),CV均小于2%(n=9);采用该法分析2个已上市产品的酶活性。结论建立了生色底物法测定rhα-GAL的酶活性,该方法灵敏度高、精密度及准确度较好,可用于rhα-GAL产品的酶活性评价和质量控制。

OBJECTIVE To establish a more sensitive,simple,accurate and stable method for determining the activity of recombinant humanα-galactosidase A(rhα-GAL)based on the kinetic theory of enzyme reaction and study the assay conditions for the activity assay.METHODS The optimum conditions of the assay system were as follows:50 mmol·L^(-1)p-nitrophenyl-α-D-galactopyranoside was used as the substrate,the concentration of the enzyme was 1.67μg·mL^(-1),the reaction was accurately carried out in a water bath at 37℃for 15 min,then the reaction was terminated by glycine buffer(pH 10.5),and the absorbance was measured at 400 nm using a microplate reader.RESULTS The method had good specificity.Rhα-GAL showed a good linear relationship with the enzymatic reaction rate in the range of 0.83-2.51 mg·mL^(-1)(r=0.9998).The recoveries of validation solutions at 50%,80%,100%,125%and 150%concentrations were in the range of 94.2%-101.8%(n=18),and the CVs of the measured results were between 2.0%and 5.5%(n=18).The CV of 12 independent tests of the same sample was 2.21%(n=12).The effects of slight changes in water bath temperature,reaction time and substrate concentration in the reaction system on the results were investigated,confirming the good robustness of the method.The reconstituted sample showed good stability when stored at 2-8℃for 48 h.p-Nitrophenol showed a good linear relationship with the absorbance in the range of 0.01-0.15 mmol·L^(-1)(r=0.9997).The recoveries of p-nitrophenol solution at five concentrations were in the range of 94.9%-105.1%(n=9),and the CVs were all below 2.0%(n=9).The activity of two rhα-GAL products was determined by this method.CONCLUSION A chromogenic substrate method was established to determine the activity of rhα-GAL and validated with good sensitivity,precision and accuracy,which can be used for the activity evaluation and quality control of the product.