Screening and optimization of method for removing free polysaccharides from type 9V pneumococcal polysaccharide-protein conjugate
10.13200/j.cnki.cjb.004228
- VernacularTitle: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
- Author:
CHEN Qian
- Publication Type:Journal Article
- Keywords:
Pneumococcus;
Polysaccharide-protein conjugate;
Free polysaccharides;
Hydrophobic interaction chromatography
- From:
Chinese Journal of Biologicals
2024;37(5):593-597
- CountryChina
- Language:Chinese
-
Abstract:
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-CRM_(197) 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 than25% 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.
