摘要
目的建立一种检测23F型肺炎链球菌多糖结合物游离蛋白含量的方法,并进行方法学验证。方法采用体积排阻高效液相色谱(size exclusion chromatography and high performance liquid chromatography,SEC-HPLC)法检测23F型肺炎链球菌多糖结合物游离蛋白含量。色谱柱:TSKgel G3000(300 mm×78 mm,5μm);流动相:10 mmol/L PBS(150 mmol/L NaCl,pH 6. 8);检测波长:214 nm;流速:0. 5 m L/min;柱温:25℃;自动进样:100μL。同时对该方法进行线性、精密度、准确度、耐用性及专属性验证。结果建立的方法在纯化载体蛋白破伤风类毒素(tetanus toxoid,TT)含量为3～18μg/mL时,与峰面积呈良好的线性关系,线性相关系数(R2)> 0. 99;23F-2017001、23F-2017002、23F-2017003批供试品重复性相对标准偏差(RSD)分别为0. 1%、0. 3%、1. 0%,中间精密性RSD分别为3. 7%、8. 5%、4. 8%;加入6、8、10μg/mL纯化载体蛋白TT样品的3次检测结果回收率在93. 2%～102. 6%之间,回收率的RSD均为0. 6%;进样温度为20～30℃条件下的保留时间和峰面积相对偏倚在-0. 1%～0. 5%之间;流动相溶液中含甘氨酸及低浓度硼酸时对检测结果无干扰,硼酸浓度为20 mmol/L以上时对游离蛋白峰检测有一定干扰。结论本实验建立的方法具有良好的线性、精密性、准确性、耐用性及专属性,可用于23F型肺炎链球菌多糖结合物游离蛋白含量检测。
Objective To develop and validate a method for determination of free protein content in type 23F pneumococcal polysaccharide conjugate. Methods The free protein content in type 23 F pneumococcal polysaccharide conjugate was determined by size exclusion chromatography and high performance liquid chromatography(SEC-HPLC).TSKgel G3000(300 mm × 78 mm,5 μm)was used serving 10 mmol/L PBS containing 150 mmol/L sodium chloride(pH 6. 8)as mobile phase at a flow rate of 0. 5 mL/min,a detection wavelength of 214 nm and a temperature of 25 ℃.The volume of sample for loading was 100 μL. The developed method was validated for linearity,precision,accuracy,durability and specificity. Results The content of purified tetanus toxoid(TT)determined by the method,at a range of 3 ~ 18 μg/mL,showed good linear relationship to the peak area,with a linear correlation coefficient(R2 value)of more than 0. 99. The RSDs of reproducibility test results of samples with lot numbers of 23 F-2017001,23 F-2017002 and 23 F-2017003 were 0. 1%,0. 3% and 1. 0%,while those of intermediate precision test results were 3. 7%,8. 5% and 4. 8%,respectively. The recoveries of samples added with 6,8 and 10 μg/mL purified TT were 93. 2%~ 102. 6%,each with a RSD of 0. 6%. The relative biases of retention time and peak area at temperature for sample loading of 20 ~ 30 ℃ were-0. 1%~ 0. 5%. The glycine and low concentration boric acid in mobile phase showed no interference to test result.However,the boric acid at a concentration of more than 20 mmol/L showed a certain interference to free protein peak.Conclusion The developed method showed good linearity,precision,accuracy,durability and specificity,which might be used for determination of free protein content in type 23 F pneumococcal polysaccharide conjugate.
作者
邓海清
尹珊珊
郝倩
顾行举
刘杨
张艳红
高宇皎
刘建凯
DENG Hai-qing;YIN Shan-shan;HAO Qian;GU Xing-ju;LIU Yang;ZHANG Yan-hong;GAO Yu-jiao;LIU Jian-kai(Center of Research and Development,Minhai Biotechnology Co.,Ltd.,Beijing 102600,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2019年第5期579-583,共5页
Chinese Journal of Biologicals
关键词
肺炎链球菌
多糖蛋白结合物
游离蛋白
体积排阻高效液相色谱
Streptococcus pneumonia
Polysaccharide and protein conjugate
Free protein
Size exclusion chromatography and high performance liquid chromatography(SEC-HPLC)
