紫外-可见分光光度法检测高纯度人C1酯酶抑制剂蛋白质含量的经验公式  

Establishment of empirical formula for determination of protein content of high purity human C1 esterase inhibitor by ultraviolet-visible spectrophotometry

作　　者：汪菲菲 李娟 陈克金 朱晨 纪德铭 王上 彭焱 李策生 胡勇 周志军 WANG Fei-fei;LI Juan;CHEN Ke-jin;ZHU Chen;JI De-ming;WANG Shang;PENG Yan;LI Ce-sheng;HU Yong;ZHOU Zhi-jun(Scientific Research and Development Department,Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]国药集团武汉血液制品有限公司科研开发部,湖北武汉430207

出　　处：《微生物学免疫学进展》2023年第3期28-35,共8页Progress In Microbiology and Immunology

摘　　要：目的建立高纯度人C1酯酶抑制剂(C1 esterase inhibitor,C1-INH)蛋白质含量(质量浓度)检测的紫外-可见光分光光度法(简称A_(280 nm)法),得到双对数方程的经验公式,用于C1-INH精纯阶段含目标蛋白样品的蛋白质含量在线检测。方法以1批高效液相色谱(high performance liquid chromatography,HPLC)纯度98.49%的C1-INH原液作为对照品,用注射用水稀释获得不同质量浓度的稀释品,分别检测稀释品A_(280 nm)值,以稀释品的A_(280 nm)值与理论质量浓度进行不同方程的线性拟合,选取回收率在理论值100%±10%、R^(2)>0.99的质量浓度区间建立最佳拟合方程。在此线性范围内,用原始值和消光值分别拟合得到不同的方程,对2种拟合方程的准确度、精密度和稳定性进行验证。通过原始值均值方程和不同基质液的检测均值来建立经验公式,用该经验公式计算不同批次纯化工艺中间样品的蛋白质质量浓度并与免疫比浊法和凯氏定氮法的检测结果进行比较,确定其准确性。用该经验公式指导C1-INH精纯样品的检测。结果建立的A_(280 nm)法采用双对数方程拟合标准曲线的线性范围为0.214~3.567 mg/mL;在此线性范围内,用原始值和消光值分别拟合双对数方程,2个标准曲线的重复性和稳定性均较好,且高、中、低值质控品及检测下限的准确度(即回收率)<100%±7%,精密度CV<3%。含目标蛋白质的多种基质液按照原始均值方程计算得到对应的蛋白质质量浓度在0.044~0.064 mg/mL,均值为0.051 mg/mL;经验公式为:蛋白质质量浓度x={10(lg(y)+0.3820.764)-0.051}×稀释倍数,用该公式计算的添加了对照品的各基质的回收率在96.636%~109.533%。检测不同批次纯化工艺中间样品的结果显示,对于精纯样品,该经验公式与凯氏定氮法的结果比值在0.93~1.12,免疫比浊法结果与凯氏定氮法的结果比值在1.19~2.33。用该经验公式计算的不同精纯阶段中间样品的结果与凯氏定Objective To establish a UVVis spectrophotometric method(A_(280 nm)method)for quantitative detection of high purity human C1esterase inhibitor(C1INH)protein content(concentrstion),and obtain an empirical formula of double logarithmic equation which can be used for rapid calculation of protein content of samples with target protein in the fine purification stage of C1INH process.Methods A batch of C1INH bulk with HPLC purity of 98.49%was used as reference substance,diluted with water for injection to obtain diluted samples with different concentrations,and their A_(280 nm)were detected.The A_(280 nm)values and theoretical concentrations of diluted samples were linearly fitted with different equations,and the best fitting equation was established in the selected concentrations range when the recovery rate’s theoretical value was 100±10%and R^(2)>0.99.In this linear range,the original value and extinction value are used to fit different equations,then the accuracy,precision and stability of the two fitting equations are validated.The empirical formula is established by establishing the original value mean equation and the detection mean value of different matrix liquids.The protein concentration of different batches of process samples was calculated by the empirical formula and compared with the results of the Kjeldahl method and immunoturbidimetry to determine its accuracy.The empirical formula was used to guide the detection of highpurity C1INH samples.Results The linear range of fitted standard curve of A_(280 nm)method is 0.214-3.567 mg/mL by using double logarithmic equation.In this linear range,the original value and extinction value are used to fit the double logarithmic equation respectively.The repeatability and stability of the two standard curves are good.The accuracy(recovery)of the high,medium and low values of quality control and the lower limit of detection were all within 100%±7%,and the precision(CV)were all within 3%.According to the original mean value equation,the corresponding protein concen

关 键 词：蛋白质含量检测的紫外-可见光分光光度法 免疫比浊法 凯氏定氮法 人C1酯酶抑制剂 线性范围 

分 类 号：R392-33[医药卫生—免疫学]