9V型肺炎球菌多糖蛋白结合物中游离多糖去除方法的筛选及优化

Screening and optimization of method for removing free polysaccharides from type 9V pneumococcal polysaccharide⁃protein conjugate

目的筛选一种有效去除9V型肺炎球菌(Pn9V)多糖蛋白结合物中游离多糖的方法,并进行工艺优化及稳定性验证,以期用于多价肺炎结合疫苗的制备。方法制备Pn9V-CRM197结合物,分别采用分子筛(Sepharose 4FF)、离子交换层析(DEAE Sepharose FF)、疏水层析(GP-Butyl)及硫酸铵(ammonium sulfate,AS)盐析法去除结合物中的游离多糖,通过比较游离多糖百分比、多糖回收率及蛋白回收率筛选最适方法,并对确定的方法进行工艺优化。连续制备3批结合物,验证工艺的稳定性。结果疏水层析和离子交换层析均可将Pn9V-CRM197结合物的游离多糖降至5%以下,疏水层析多糖及蛋白回收率(34.7%和50.1%)高于离子交换层析(16.2%和25.7%),选择疏水层析去除结合物中的游离多糖。优化的工艺为1.1 mol/L AS上样,注射用水洗脱,层析载量为1.81 mg蛋白/mL填料。连续制备的3批结合物游离多糖去除率、多糖回收率及蛋白回收率良好,且批间一致性高;2~8℃放置6个月,结合物稳定性良好,(25±2)℃放置6个月,游离多糖百分比显著增加(F=5.83 e-32,P=0.003),且>25%,不适宜高温保存。结论建立了有效去除Pn9V蛋白结合物中游离多糖的疏水层析工艺,该工艺稳定性良好,也为其他型别肺炎球菌多糖蛋白结合物游离多糖的去除提供了参考。

Objective To screen an effective method for removing free polysaccharides in polysaccharide-protein conjugate of pneumococcus type 9V(Pn9V),and to optimize the process and verify its stability,so as to use it in the preparation of multivalent conjugate vaccine against pneumococcus.Methods Pn9V-CRM197 conjugate was prepared,of which the free polysaccharides were removed by molecular sieve(Sepharose 4FF),ion exchange chromatography(DEAE Sepharose FF),hydrophobic chromatography(GP-Butyl)and ammonium sulfate(AS)salting out.The optimal method was selected by comparing the percentage of free polysaccharides,the recovery rate of polysaccharides and the recovery rate of proteins,and the determined method was optimized for the process.Three batches of conjugates were prepared continuously to verify the stability of the process.Results Both hydrophobic chromatography and ion exchange chromatography reduced the free polysaccharides in Pn9V-CRM197 conjugate to less than 5%.The recovery rates of polysaccharide and protein by hydrophobic chromatography were 34.7%and 50.1%,respectively,which were higher than those by ion exchange chromatography(16.2%and 25.7%).Hydrophobic chromatography was determined with the optimized process including 1.1 mol/L AS as the loading buffer,water for injection used for elution,and the conjugate loading of 1.81 mg protein/mL filler.The free polysaccharide removal rate,polysaccharide recovery rate and protein recovery rate of the conjugates prepared continuously in three batches were good,and the consistency among batches was high.The conjugates had good stability when stored at 2-8℃for 6 months,and the percentage of free polysaccharides increased significantly(F=5.83 e-32,P=0.003)and was more than 25%when stored at(25±2)℃for 6 months,indicating that high temperature storage was not suitable for the conjugates.Conclusion A hydrophobic chromatography process was established to effectively remove free polysaccharides from Pn9V polysaccharide-protein conjugate,which has good stability and provides a reference for the removal of free polysaccharides in other pneumococcal polysaccharide-protein conjugates.