摘要
目的建立人C1酯酶抑制剂(C1 esterase inhibitor or C1 inhibitor,C1-INH)活性的动态显色检测方法,并进行验证。方法采用棋盘法分析C1酯酶与发色底物C1E 5603[CH3OC-Lys(Cbo)-Gly-Arg-pNA·AcOH]反应后的A值变化率(ΔA/min),确定能使不同浓度酶饱和的最低底物浓度。将C1-INH浓缩制剂标准品与C1酯酶孵育,剩余的C1酯酶与底物C1E 5603作用产生A值,将ΔA/min与C1-INH活性进行拟合,绘制标准曲线。验证该方法的线性范围、选择性、特异性、准确度、精密度、稳定性,并进行样品添加试验。将验证后的该方法应用于C1-INH制备工艺过程样品的分析。结果确定C1酯酶的浓度为28μg/mL,底物浓度为2.4 mmol/L,反应结果监测4 min。C1-INH标准品在0.15~0.025 IU/m 范围内与ΔA/min呈良好的线性关系,R2≥0.99,校正标样回算浓度在标示值的90.2%~105.4%内;CM阳离子交换层析洗脱缓冲液、HIC疏水层析平衡缓冲液、人纤溶酶原(plasminogen,Plg)、人纤溶酶(plasmin,Plm)的响应值均低于定量下限响应的7.2%,并低于内标响应的2.7%;20 mmol/L以下的pH 5.0Na Ac及300 mmol/L以下的Na Cl对C1-INH活性检测无影响;检测定量上限、高、中、低、定量下限质控样品,批内准确度在90.0%~114.8%之间,批内CV在0.7%~13.7%之间,批间准确度在94.2%~108.3%之间,批间CV在1.2%~9.7%之间;C1酯酶与底物C1E 5603室温放置3 h不影响检测结果;样品添加试验回收率在94.5%~106.1%之间。CM阳离子交换层析洗脱液、DEAE阴离子交换层析洗脱液、HIC疏水层析流穿液中C1酯酶抑制剂的特异性活性逐渐升高,分别为0.79、1.84、2.61 IU/g。结论本方法具有良好的选择性、准确度、精密度、特异性及稳定性,可作为C1-INH制备工艺中的内部控制指标。
Objective To develop and verify a dynamic chromogenic method for determination of activity of C1 esterase inhibitor（C1-INH）. Methods The change of absorbance value（ΔA/min） of C1 esterase reacting with chromogenic substrate C1E 5603 [（CH3OC-Lys）（Cbo）-Gly-Arg-pNA·AcOH]was analyzed by checkerboard method to determine the minimum substrate concentrations saturating the esterase at various concentrations. Concentrated C1-INH standard was incubated with C1 esterase, and the remained C1 esterase was reacted with substrate to produce absorbance value. A standard curve was established with ΔA/min fitting with the activity of C1-INH standard. The dynamic chromogenic assay was verified for linear range, selectivity, specificity, accuracy, precision and stability, subjected to sample addition test,and used for analysis of samples in the preparation process of C1-INH. Results The C1 esterase concentration was determined as 28 μg/mL, while the substrate concentration was 2. 4 mmol/L, and the ΔA/min was monitored for4 min. The linear range of the standard curve of C1-INH was 0. 15 - 0. 025 IU/mL. The recovery of calibration standard was 90. 2% - 105. 4%, with a R2 value of not less than 0. 99. The ΔA/min of CM cation exchange chromatography elution buffer, HIC hydrophobic chromatography equilibrium buffer, human plasminogen（Plg） and plasmin（Plm） were lower than 7. 2% of that of samples at a concentration of lower limit of range（LOR）and 2. 7% of that of internal standard.The concentration of pH 5. 0 NaAc less than 20 mmol/L and NaCl less than 300 mmol/L showed no interference to the activity assay. The recoveries of quality control samples at a concentration of higher LOR, high, moderate, low and lower LOR were 90. 0% - 114. 8% in intra-assay and 94. 2% - 108. 3% in inter-assay, with coefficients of variation（CVs） of 0. 7% - 13. 7% and 1. 2% - 9. 7% respectively. Keeping the C1 esterase and substrate C1E at room temperature for 3 h showed no interference to the activity assay. The recovery rate of sample addition test was 94. 5% - 106. 1%. The specific activity in C1-INH of CM cation exchange chromatography elution buffer, DEAE anion exchange chromatography elution buffer and HIC hydrophobic chromatography flow through liquid were 0. 79, 1. 84 and 2. 61 IU/g respectively.Conclusion The method showed good selectiviy, accuracy, precision, specificity and stability, which might be used as an indicator of internal quality control in preparation process of C1-INH.
作者
汪菲菲
岳胜兰
周志军
彭焱
李娟
张云骁
李策生
胡勇
WANG Fei-fei;YUE Sheng-lan;ZHOU Zhi-jun;PENG Yan;LI Juan;ZANG Yun-xiao;LI Ce-sheng;HU Yong(Department of Blood Products Laboratory,Sinopharm Wuhan Plasma-derived Biatherapies Co.,Ltd.,Wuhan 430207,Hubei Province,Chin)
出处
《中国生物制品学杂志》
CAS
CSCD
2018年第7期751-757,共7页
Chinese Journal of Biologicals
基金
2013年湖北省重大科技创新计划(2013ACC001)
武汉国际科技合作计划(2015030809020360)
关键词
C1酯酶抑制剂
动态显色法
活性
C1 esterase inhibitor
Dynamic chromogenic assay
Activity
