肺炎球菌多糖-蛋白结合疫苗中4-二甲氨基吡啶残留量高效液相色谱检测方法的建立及验证  

Development and verification of high performance liquid chromatography for determination of residual 4-dimethylaminopyridine content in pneumococcal polysaccharide-protein conjugate vaccine

作　　者：李双花 赵萍 田洪敏 李红 董威 陈煜 鲍路伟 吴克 LI Shuang-hua;ZHAO Ping;TIAN Hong-min;LI Hong;DONG Wei;CHEN Yu;BAO Lu-wei;WU Ke(Hubei Provincial Engineering Laboratory of Novel Vaccine&Recombinant Protein,Wuhan Bravovax Co.,Ltd.,Wuhan 430075,Hubei Province,China)

机构地区：[1]武汉博沃生物科技有限公司技术中心新型疫苗与重组蛋白湖北省工程实验室,湖北武汉430075

出　　处：《中国生物制品学杂志》2023年第2期211-216,共6页Chinese Journal of Biologicals

基　　金：湖北省2016年度“双创战略团队”(鄂科技通[2017]36号)。

摘　　要：目的建立肺炎球菌多糖-蛋白结合疫苗中4-二甲氨基吡啶(4-dimethylaminopyridine,DMAP)含量检测的高效液相色谱(HPLC)法,并对该方法进行验证。方法通过对色谱柱类型、流动相组成的优化建立检测肺炎球菌多糖-CRM197蛋白结合疫苗中DMAP残留量的HPLC法,对该方法进行专属性、准确度、重复性和重现性验证,并确定检出限、定量限及线性范围。用建立的方法检测13种不同血清型的肺炎球菌多糖-CRM197蛋白结合疫苗及1-氰基-4-二甲氨基吡啶四氟化硼(1-cyano-4-dimethylamino pyridinium tetrafluoroborate,CDAP)活化肺炎球菌多糖中间产物中DMAP残留量。结果优化后的色谱条件:色谱柱为Sepax HP-C18(5μm,4.6 mm×250 mm);流动相为5 mmol/L庚烷磺酸钠溶液(含20 mmol/L磷酸二氢钾,磷酸调pH至3.0)∶乙腈=87∶13(V/V);检测波长为280 nm;流速为1 mL/min;进样体积为10μL;柱温为35℃。该方法不受供试品机制和前处理溶剂的干扰,专属性好;检出限和定量限分别为3.6和14.4 ng/mL,在0.05~10μg/mL范围内线性良好,R2=0.9999;加标回收率为83%~105%,重复性RSD为0.28%~0.72%,重现性RSD为7.38%。13种不同血清型肺炎球菌多糖-CRM197蛋白结合疫苗中DMAP残留量为0.135~1.635μg/mL;CDAP活化肺炎球菌多糖第一步中间产物即可检测出DMAP,未检测到CDAP。结论建立的HPLC法操作简便,专属性、准确度、重复性和重现性均较好,可作为肺炎球菌多糖-蛋白结合疫苗质量控制的有效检测方法。Objective To develop and verify a high performance liquid chromatography(HPLC)for determination of residual 4-dimethylamino-pyridine(DMAP)content in pneumococcal polysaccharide-protein conjugate vaccine.Methods A HPLC method for determination of residual DMAP content in pneumococcal polysaccharide-CRM197 protein conjugate vaccine was developed by optimization of type of chromatographic column and composition of mobile phase,verified for specificity,accuracy,repeatability and reproducibility,and determined for limit of detection(LOD),limit of quantitation(LOQ)and linear range.The residual DMAP contents in pneumococcal polysaccharide-CRM197 protein conjugate vaccine of 13 serotypes and the intermediates of 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP)-activated pneumococcal polysaccharide were determined by the developed method.Results The condition for HPLC was optimized as follows:Sepax HP-C18 chromatographic column(5μm,4.6 mm×250 mm)was adopted,using the mixture of 5 mmol/L sodium heptanesulfonate containing 20 mmol/L potassium dihydrogen phosphate(the pH value was adjusted to 3.0 with phosphoric acid)and acetonitrile at a ratio of 87∶13(v/v)as mobile phase at a detection wavelength of 280 nm,a flow rate of 1 mL/min,a sample load of 10μL and a column temperature of 35℃.The mechanism of samples and solvent for pre-treatment showed no interference to the determination result,indicating good specificity of the developed method.The LOD and LOQ were 3.6 and 14.4 ng/mL respectively,while the linear range was 0.05~10μg/mL with a R2value of0.9999.The spike recovery rate was 83%~105%,while the RSDs in repeatability and reproducibility tests were 0.28%~0.72%and 7.38%respectively.The residual DMAP contents in pneumococcal polysaccharide-CRM197 protein conjugate vaccine of 13 serotypes were 0.135~1.635μg/mL.DMAP was detected in the first step of activation of pneumococcal polysaccharide with CDAP,while no CDAP was detected.Conclusion The developed HPLC method is simple,specific,accurate,repeatable and

关 键 词：肺炎球菌多糖 蛋白 结合疫苗 4-二甲氨基吡啶 高效液相色谱法 

分 类 号：R392-33[医药卫生—免疫学]