A群脑膜炎球菌多糖结合物原液中游离糖含量测定方法的建立及其验证

Development and validation of a determination method for free polysaccharide content in bulk of group A meningococcal polysaccharide conjugate

目的 建立A群脑膜炎球菌多糖结合物原液中游离糖含量测定的方法,并进行验证。方法 采用脱氧胆酸钠（sodium deoxycholate,DOC）法,分别取1 ml 10、50、100、150、200、250、294μg/ml载体蛋白破伤风类毒素（tetanus toxoid,TT）,与100μl 1%DOC溶液（pH 7.3）混匀,离心收集上清,检测蛋白含量,确定DOC沉淀载体蛋白范围;用不同离子强度梯度的缓冲液（0.2、0.4、0.6、0.8、1.0 mol/L氯化钠）稀释游离多糖与载体蛋白混合物溶液,收集上清,检测多糖含量,确定游离多糖分离条件;以1%DOC溶液作为稀释剂绘制参考品磷含量测定标准曲线,确定DOC存在对磷含量测定结果的影响,建立DOC沉淀蛋白结合磷含量测定方法测定A群脑膜炎球菌多糖结合物中游离糖含量的方法,并对建立的方法进行精密度、准确性及重复性验证。结果DOC沉淀载体蛋白浓度控制在250μg/ml以下;将0.8 mol/L氯化钠溶液作为A群脑膜炎球菌多糖结合物中游离糖含量测定所需最佳稀释液;以1%DOC溶液作为稀释剂绘制的磷含量标准曲线线性范围在0～4μg/ml时,r2为0.999 9。A群脑膜炎球菌多糖结合物原液及游离糖对照品与载体蛋白混合物沉淀中蛋白回收率在95%～110%之间;游离糖添加试验回收率均在95%以上;4批A群脑膜炎球菌多糖蛋白结合物游离糖含量平均值均在规定范围内。结论 建立的DOC沉淀蛋白结合磷含量测定方法可有效分离结合物与游离多糖,并准确测定结合物含量。

Objective To develop and validate a method for determination of free polysaccharide in bulk of group A meningococcal polysaccharide（GAMP）conjugate. Methods A portion of 1 ml of tetanus toxoid（TT）as carrier protein,at concentrations of 1,10,50,100,150,200,250 and 294 μg / ml respectively,were mixed with 100 μl 1% sodium deoxycholate（DOC）（pH 7. 3）,and the supernatants were collected by centrifugation and determined for protein content,based on which the range of carrier protein concentration precipitated with DOC was determined. The mixtures of free polysaccharide and carrier protein were diluted with buffers at various ionic strengths（0. 2,0. 4,0. 6,0. 8 and 1. 0 mol / L）sodium chloride,of which the supernatants were collected and tested for polysaccharide content to determine the condition for separation of free polysaccharide. The standard curve for determination of phosphor content was plotted using 1%DOC as a diluent to evaluate the presence of DOC on determination result of phosphorus content. DOC precipitation combined with phosphor content determination method for free polysaccharide content in GAMP conjugate was developed,and verified for precision,accuarcy and reproducibility. Results The carrier protein concentration for DOC precipitation was controlled at less than 250 μg / ml. Sodium chloride at a concentration of 0. 8 mol / L was served as the diluent for determination of free polysaccharide in GAMP conjugate. The linear range of standard curve for determination of phosphorcontent plotted using 1% DOC as a diluent was 0 ~ 4 μg / ml,with a r2 value of 0. 999 9. The recovery rates of proteins in bulk precipitate of GAMP and mixture precipitate of free polysaccharide and carrier protein were 95% ~ 110%. All the recovery rates of free polysaccharide in spiking study were more than 95%. The mean free polysaccharide contents in four batches of GAMP were within the range specified. Conclusion The developed DOC precipitation combined with phosphor determination method was effective for separation of conjugate from free polysaccharide and accurate determination of conjugate content.