反相高效液相色谱法检测白喉毒素无毒突变体CRM197蛋白纯度  

Determination of purity of nontoxic mutant CRM197 of diphtheria toxin by reversed-phase high performance liquid chromatography

作　　者：何刚 黄杰 孙俊 魏鑫 冯树宏 侯文礼 HE Gang;HUANG Jie;SUN Jun;WEI Xin;FENG Shu-hong;HOU Wen-li(Research and Development Center,Chengdu Kanghua Biological Products Co.,Ltd.,Chengdu 611130,Sichuan Province,China)

机构地区：[1]成都康华生物制品股份有限公司研发中心,四川成都611130

出　　处：《微生物学免疫学进展》2023年第2期34-40,共7页Progress In Microbiology and Immunology

基　　金：四川省科技创新人才项目(2022JDRC0048)。

摘　　要：目的 建立反相高效液相色谱法(reverse phase high performance liquid chromatography, RP-HPLC)检测白喉毒素无毒突变体CRM197蛋白纯度。方法 利用Agilent AdvanceBio RP-mAb SB-C8(100 mm×2.1 mm)分析柱和Agilent1260高效液相色谱系统,以含0.1%三氟乙酸水溶液-异丙醇(98∶2)为流动相A,以含0.1%三氟乙酸乙腈溶液为流动相B,进行梯度洗脱,体积流速为0.5 mL/min,检测波长为280 nm,柱温为65℃,进样体积为10μL,采用面积归一法检测CRM197蛋白纯度,并对方法的适用性、专属性、重复性、中间精密度、线性、灵敏度和耐用性指标进行考察。用建立的方法检测CRM197蛋白酸处理供试品溶液、碱处理供试品溶液及3批原液的纯度。结果 建立的方法系统适用性良好;专属性验证表明空白溶液在目标峰积分范围内无干扰峰,热处理CRM197蛋白目标峰与其他杂质峰分离度>1.5;6次重复进样目标峰面积相对标准偏差(relative standard deviation,RSD)为1.14%,保留时间RSD为0.81%;中间精密度分析目标峰面积RSD为1.62%,保留时间RSD为0.06%;CRM197蛋白含量在20~2 000μg/mL范围内,与峰面积存在良好的线性关系(R^(2)=0.999 8);最低检测限为10μg/mL,定量限为20μg/mL;在改变柱温色谱条件时,目标峰面积百分比与保留时间均RSD≤5.00%,耐用性研究合格。对酸处理、碱处理供试品溶液及3批原液进行纯度检测,纯度在76.5%~96.9%,结果准确。结论 建立的RP-HPLC检测CRM197蛋白纯度的方法,经验证各项指标均符合要求,可用于CRM197蛋白的纯度检测。Objective To establish a reverse phase high performance liquid chromatography(RP-HPLC) method for detecting the purity of diphtheria toxin non-toxic mutant CRM197 protein. Methods Using Agilent AdvanceBio RP-mAb SB-C8 analytical column(100 mm×2.1 mm) and HPLC system(Agilent 1260). with 0.1% trifluoroacetic acid aqueous solution-isopropanol(98:2) as mobile phase A, acetonitrile solution containing 0.1% trifluoroacetic acid as mobile phase B, gradient elution was performed at the flow rate of 0.5 mL/min, the detection wavelength was 280 nm, column temperature was 65 ℃,and the injection volume was 10 μL. The purity of CRM197 protein was determined by peak area normalization method. This assay was subsequently validated for its system applicability, specificity, repeatability, intermediate precision and linearity, and determined limit of detection(LOD) and limit of quantitation(LOQ), and durability. The established method was used to assay the purity of the acid-treated test solution, the alkali-treated test solution and three batches of stock solution of CRM197 protein. Results The established RP-HPLC method showed good systemic suitability. Specificity validation showed that there was no interference peak in the blank solvent;the separation of the target peak of CRM197 protein from other impurity peaks was >1.5. While the relative standard deviations(RSD) value of peak area and retention time in six tests were 1.14% and 0.81%, respectively. The RSD of peak area and retention time were 1.62% and 0.06%, respectively in the intermediate precision analysis. The CRM197 protein content had a good linear relationship with the peak area in the range of 20-2 000 μg/mL(R^(2)=0.999 8). The LOD was 10 μg/mL, and the LOQ was 20 μg/mL. When the column temperature chromatographic conditions were changed, the percentage of the target peak area and the retention time were the same. The target peak area percentage and retention time were both RSD≤5.00%. The purity of the acid-treated and alkali-treated test solutions and th

关 键 词：白喉毒素 无毒突变体 CRM197蛋白 纯度 反相高效液相色谱法 结合疫苗 

分 类 号：R378[医药卫生—病原生物学]