聚乙二醇化人睫状神经营养因子蛋白含量反相高效液相色谱检测方法的建立及验证  

Development and validation of a reverse-phase high performance liquid chromatography for determination of protein content of PEGylated human ciliary neurotrophic factor

作　　者：刘建伟 李素贞 柳森 王健 LIU Jian-wei;LI Su-zhen;LIU Sen;WANG Jian(National Vaccine and Serum Institute,Bejing 101111,China)

机构地区：[1]国药中生生物技术研究院有限公司,北京101111

出　　处：《中国生物制品学杂志》2020年第10期1156-1160,共5页Chinese Journal of Biologicals

基　　金：北京市科委“生物医药与生命科学创新培育研究-创新品种临床前研究专项”(z151100003915068);国药集团新产品开发基金(2011SW02)。

摘　　要：目的建立聚乙二醇化重组人睫状神经营养因子(PEGylated ciliary neurotrophic factor,PEG-CNTF)蛋白含量反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)检测方法,并进行验证。方法以未修饰的人CNTF原型蛋白为对照品,采用反相高效液相色谱法(reversed-phase high performance liquid chromatography,RP-HPLC)测定PEG-CNTF的蛋白含量。色谱柱:Jupiter 5u C4300A(150 mm×4.6 mm);流动相:0.1%三氟乙酸的水溶液、0.1%三氟乙酸的乙腈;检测波长:280 nm;流速:1 mL/min;柱温:室温;进样体积:20μL。对该方法进行专属性、线性、精密性、准确性、耐用性验证。结果空白溶剂在CNTF、PEG-CNTF样品设定积分范围内无特异峰出现;建立的方法在PEG-CNTF含量为0.125~4 mg/mL浓度范围内线性关系良好(R2>0.99);供试品重复性检测的相对标准偏差(RSD)分别为0.21%、0.19%、0.45%,中间精密性检测的RSD分别为0.42%、0.59%、0.83%;制备的高、中、低3个浓度供试品检测结果的回收率均在95%~101%之间;该方法的定量限为50μg/mL,检测限为15.625μg/mL;耐用性验证中连续5次进样CNTF、PEG-CNTF峰面积及保留时间的RSD均小于2%。结论建立的方法具有良好的专属性、线性、精密性、准确性、耐用性,可用于PEG-CNTF中蛋白含量的检测。Objective To develop and validate a method for determination of protein content of PEGylated human ciliary neurotrophic factor(PEG-CNTF)by reverse-phase high performance liquid chromatography.Methods The protein content of PEG-CNTF was determined by RP-HPLC using the unmodified human CNTF prototype monomer protein as control.Jupiter 5 u C4300 A(150 mm×4.6 mm)gel column was adopted serving 0.1%trifluoroacetic acid aqueous solution and 0.1%trifluoroacetic acid in acetonitrile as mobile phase at a flow rate of 1 mL/min,a column temperature of room temperature and a detection wavelength of 280 nm.The sample load was 20μL.The developed methed was validated for specificity,linearity,precision,accuracy and durability.Results The blank control sample showed no specific peak in the defined integration range of CNTF and PEG-CNTF.The PEG-CNTF concentration at a range of 0.125~4 mg/mL showed good relationship to the peak area(R2>0.99).The RSDs of reproducibility test results of three batches samples were 0.21%,0.19%and 0.41%,while those of intermediate precision test results were 0.42%,0.59%and 0.83%,respectively.The recovery rates of prepared test samples at high,moderate and low concentrations were between 95%and 101%.The limit of quantitation(LOQ)and limit of detection(LOD)of the developed method were 50 and 15.625μg/mL respectively.In the durability test,the RSDs of retention time and peak area of CNTF and PEG-CNTF in five consecutive sample loadings were less t han 2%.Conclusion The developed method showed good specificity,linearity,precision,accuracy and durability.The method may be used for determination of the protein content of PEG-CNTF.

关 键 词：聚乙二醇化重组人睫状神经营养因子 蛋白含量 反相高效液相色谱 

分 类 号：O657.7[理学—分析化学]