高效分子排阻色谱法检测冻干b型流感嗜血杆菌结合疫苗原液游离载体蛋白含量方法的建立与验证  

Development and validation of a HPSEC method for determination of free protein content in bulk of freeze-dried Haemophilus influenzae type b polysaccharide conjugate vaccine

作　　者：马平 郑喆 王栋 蒋奕 喻大鑫 丁文博 安建科 杜送田 马国荣 MA Ping;ZHENG Zhe;WANG Dong;JIANG Yi;YU Daxing;DING Wenbo;AN Jianke;DU Songtian;MA Guorong(The Third Bacterial Vaccine Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司菌苗三室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2023年第5期39-46,共8页Progress In Microbiology and Immunology

摘　　要：目的 建立冻干b型流感嗜血杆菌结合疫苗原液中游离载体蛋白含量的高效分子排阻色谱(high performance size exclusion chromatography,HPSEC)检测方法,并对其进行方法学验证。方法 采用HPSEC法,通过对TT对照品出峰时间的测定,对分离效果和流速的考察,建立HPSEC检测游离载体蛋白含量的方法,确认色谱条件为:色谱柱:TSKgel G3000SW_(XL)(7.8 mm I.D.×30 cm,5μm);流动相为0.85%氯化钠溶液;进样体积100μL;检测波长280 nm;柱温35℃。对该方法进行专属性、分离度、线性和范围、重复性、准确度和耐用性验证并确定该方法的定量限和检测限。结果 建立的方法显示对照品TT的出峰时间为17.295 min,分离度良好,确定0.5 mL/min作为样品上样流速,并对该方法进行验证。该方法检测游离蛋白的专属性、分离度良好;对照品(纯化的载体蛋白破伤风类毒素tetanus toxoid,TT)在3~36μg/mL范围内,与峰面积的线性关系良好(R^(2)>0.998 0);3批疫苗原液重复性检测游离蛋白峰面积均<1%;加入5、10、20μg/mL TT对照品溶液的添加回收率在98.0%~102.5%,RSD均<2.02%;柱温在35℃和40℃时各样品中游离蛋白含量、分离度、拖尾因子无明显差别;确定方法检测限为0.75μg/mL,定量限为2.1μg/mL。结论 建立的HPSEC法灵敏度高、操作简便、准确度高,可用于冻干b型流感嗜血杆菌结合疫苗原液中游离蛋白含量的检测。Objective To develop and verify a high performance size exclusion chromatography(HPSEC) method to determine free protein content in bulk of freeze-dried Haemophilus influenzae type b polysaccharide conjugate vaccine.Methods The free protein content in bulk of freeze-dried Haemophilus influenzae type b polysaccharide conjugate vaccine was determined by HPSEC.HPSEC method was established to determine free protein content in bulk of freeze-dried through the measurement of the peak time of purified tetanus toxoid(TT) as well as the investigation of the resolution and flow rate.TSKgel G3000SW_(XL)(7.8 mm I.D.×30 cm,5 μm) was used as solid phase serving 0.85% sodium chloride solution with detection wavelength 280 nm and coloum temperature at 35℃.The volume of sample for loading was 100 μL.Specificity,resolution,linearity,precision,accuracy,robustness and determined for quantitative and detection limits of the method were verified.Results The results showed that the peak time of TT was 17.295 min,and the separation was good.0.5 mL/min was determined as the flow rate of sample.The method which was verified showed high specificity and separation degree in determination of free protein content in polysaccharide conjugate vaccine.The result of TT content showed good linear relationship to the peak area at a range 3-36 μg/mL,with a linear correlation coefficient(R^(2)> value) more than 0.998 0.The precision detections in peak area of free protein content were less than 1% in three batches of bulks.The recoveries of samples added with 5、10、20 μg/mL purified TT were from 98.0% to 102.5%,with RSDs less than 2.02%.There were no significant differences between 35 ℃and 40 ℃ on the determinations of free protein content,resolution and tailing factor.The detection limit and quantitative limit of the method were 0.75 and 2.1 μg/mL respectively.Conclusion The developed HPSEC method showed available sensitive,simple and accuracy,which might be used for determination of free protein content in bulk of freeze-dried Haemop

关 键 词：B型流感嗜血杆菌结合疫苗 高效分子排阻色谱法 游离蛋白 含量测定 方法验证 

分 类 号：R563.1[医药卫生—呼吸系统]