论文部分内容阅读
目的优化Y群脑膜炎球菌多糖活化工艺,为A、C、W135、Y群脑膜炎球菌结合疫苗的研制奠定基础。方法分别以多糖衍化率和游离多糖含量为指标,按照三因素三水平正交试验,以不同的pH值、活化时间和活化剂加量对多糖进行活化后,与破伤风类毒素(TT)偶联,并经Sepharose4FF层析纯化后,对多糖-TT结合物进行各项生化检定、免疫原性检测及稳定性观察,确定最佳活化工艺,并对优化的工艺进行验证。结果以多糖衍化率为指标,可确定pH值和活化剂加量二因素对Y群脑膜炎球菌多糖的活化有显著影响(P<0.05),而以游离多糖为指标时,无法确定三因素对多糖的活化有显著影响(P>0.05),结合各项指标检测及稳定性观察结果 ,确定多糖活化最佳工艺为:在pH12条件下,向每mg多糖中加入等量活化剂,反应10min。采用优化的多糖活化工艺制备3批Y群脑膜炎球菌多糖-TT结合物,各项指标均符合多糖结合疫苗的常规质控标准。结论优化的多糖活化工艺可制备出游离多糖含量低、免疫原性高的Y群脑膜炎球菌多糖蛋白结合物,结果具有重复性。
Objective To optimize the activation of meningococcal polysaccharide Y in group Y, and lay a foundation for the development of meningococcal A, C, W135 and Y group conjugate vaccines. Methods Based on polysaccharide derivatization rate and free polysaccharide content, respectively, polysaccharide was activated by different pH value, activation time and activator dosage according to three factors and three levels orthogonal experiment, After purification by Sepharose4FF chromatography, the biochemical test, immunogenicity test and stability observation of the polysaccharide-TT conjugate were carried out to determine the optimal activation process and to verify the optimized process. Results The polysaccharide derivatization rate was used as an index to determine the effect of pH and activator dosage on the activation of serogroup Y meningococcal polysaccharide (P <0.05). However, when free polysaccharide was used as index, three factors Polysaccharide activation (P> 0.05), combined with the detection of various indicators and the stability of the observation results, to determine the optimum polysaccharide activation process: at pH12 conditions, adding an equal amount of activator per mg of polysaccharide, the reaction 10min. Three batches of Y meningococcal polysaccharide-TT conjugate were prepared by the optimized polysaccharide activation process. All the indexes were in accordance with the conventional quality control standards of the polysaccharide conjugate vaccine. Conclusion The optimized polysaccharide activation process can produce Y meningococcal polysaccharide protein conjugates with low free polysaccharide content and high immunogenicity, and the results are reproducible.