Y群脑膜炎球菌结合物制备过程中荚膜多糖水解条件的优化

Optimization of hydrolysis conditions of capsular polysaccharide during preparation of serogroup Y meningococcal conjugate

目的对Y群脑膜炎球菌结合物制备过程中荚膜多糖水解条件进行优化,确定水解反应参数,以适用于后续结合物制备。方法分别使用双氧水(H2O2)和冰醋酸2种水解反应试剂,在不同浓度(H2O2分别至终浓度1%和2%,冰醋酸分别至终浓度0.05和0.1 mol/L)、不同温度(37和50℃)下反应不同时间,比较水解物的分子大小;采用确定的工艺参数连续制备3批多糖水解物,检测分子大小、O-乙酰基含量,并进行结构确认和抗原性分析。结果确定了Y群脑膜炎球菌多糖水解反应参数为:水解试剂冰醋酸,水解反应温度37℃,冰醋酸终浓度0.05 mol/L,反应时间3 h。采用以上反应参数制备的3批多糖水解物在结构、特异基团及抗原性等方面完好地保留了多糖的特性,且分子大小适宜、批件一致性良好。结论优化的Y群脑膜炎球菌荚膜多糖水解反应参数可应用于多糖水解物制备,为后续稳定制备合格结合物提供了保障。

Objective To optimize the efficiency of hydrolysis of capsular polysaccharides during preparation of group Y meningococcal conjugates,and to determine the reaction parameters of hydrolysis,in order to apply them to the subsequent conjugate preparation.Methods The group Y meningococcal capsular polysaccharide was hydrolyzed under different conditions using hydrogen peroxide(H2O2)and glacial acetic acid as hydrolysis reagents at different concentrations and temperature for different time,and the molecular size of polysaccharide hydrolysates was detected.Three batches of polysaccharide hydrolysates were prepared under the optimized conditions,and were tested for molecular size,structure confirmation,O-acetyl group levels and polysaccharide antigenicity.Results The optimized conditions for hydrolysis of group Y meningococcal capsular polysaccharide were as follows:0.05 mol/L glacial acetic acid,37℃,and 3 h.Polysaccharide hydrolysates prepared under the optimized conditions maintained polysaccharide characteristics in structure,group specificity,and antigenicity,and were in proper molecular sizes.The consistency between different batches was good.Conclusion The optimized conditions for the hydrolysis of group Y meningococcal capsular polysaccharide is suitable for the preparation of polysaccharide hydrolysate,which will ensure the stable quality for subsequent conjugate preparation.