重组人溶菌酶活性检测方法研究

The study for activity detection method of recombinant human lysozyme

目的:本研究旨在以分光光度法为基础,建立重组人溶菌酶原料药和重组人溶菌酶滴眼液的活性检测方法,并对其进行方法学研究。用于重组人溶菌酶原料药和滴眼液的质量控制。方法:依据卫生部药品标准,采用分光光度法,对重组人溶菌酶活性检测方法的专属性、耐用性、精密度、线性范围以及准确性进行了验证。通过对重组人溶菌酶滴眼液进行同一时间不同样品不同底物,不同时间不同样品不同底物活性测定,确定重组人溶菌酶滴眼液的活性范围。结果:实验结果显示该活性检测方法专属性、耐用性、重复性、中间精密度、线性、准确度均良好。48 h耐用性(标准偏差为1 394. 4,RSD%为0. 95%);重复性(标准偏差为1 472. 0,RSD%为0. 99%),中间精密度(标准偏差1 000. 0,RSD%为0. 68%);检测下限为0. 625μg·m L^(-1),在5～25μg·m L^(-1)浓度范围内线性(标准曲线为y=0. 007 7x+0. 068 5,R^2=0. 991 1);准确性(回收率100. 7%～109. 6%,标准偏差为0. 032 2,RSD%为3. 05%);连续3批重组人溶菌酶原料活性分别为152 500,153 500和150 800 U·mg^(-1); 3批重组人溶菌酶滴眼液活性分别为142 000,140 000和133 000 U·mg^(-1)。连续3批重组人溶菌酶冻干粉和3批重组人溶菌酶滴眼液活性均稳定。结论:本方法可用于重组人溶菌酶原料药及重组人溶菌酶滴眼液的生物学活性测定。

Objective:To establish and validate a method for detecting the activity of recombinant human lysozyme base on spectrophotometry,and study its methodology.It could be used for the quality control of lysozyme active pharmaceutical ingredient(API)and eye drops product.Methods:Using spectrophotometry,the specificity,durability,precision,linear range and accuracy of the recombinant human lysozyme activity detection method were verified according to drug standards of China.The activity range of recombinant human lysozyme eye drops was determined through the lysozyme eye drops in the same time different samples of different substrates and different time different samples of different substrate activity determination.Results:The result showed that the specificity,durability,intermediate precision,linearity and accuracy were good.Durability during 48 h(standard deviation1 394.4,RSD%0.95%),repeatability(standard deviation 1 472,RSD%0.99%),intermediate precision(standard deviation 1 000,RSD%0.68%);the detection limit is 0.625μg·m L-1,concentration from 5 to 25μg·m L-1,linearity(standard curve y=0.007 7 x+0.068 5,R2=0.991 1);accuracy(recovery rate 100.7%~109.6%,standard deviation 0.032 2,RSD%3.05%);The activities of 3 batches of recombinant lysozyme freeze-dried powder were 152 500,153 500 and 150 800 U·mg-1 respectively,the activities of 3 batches of recombinant lysozyme eye drops were 142 000,140 000 and 133 000 U·mg-1 respectively.The activities of 3 batches of the recombinant lysozyme API and 3 batches of lysozyme eye drops were steady.Conclusion:This method could be used for the biological activity determination of recombinant lysozyme API and human recombinant lysozyme eye drops.