【摘要】 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。更多还原

【Abstract】 Objective To optimize the procedure for activation of group Y meningococcal polysaccharide and lay a foundation of preparation of groups A,C,W135 and Y meningococcal combined vaccine.Methods An orthogonal test with three factors at three levels was designed using polysaccharide derivation rate and free polysaccharide content as indicators.Polysaccharide was activated at various pH values for various minutes,with various amounts of activating agent added,then conjugated to TT.The polysaccharide-TT conjugate was purified by Sepharose 4FF chromatography,then subjected to biochemical assay,determined for immunogenicity and observed for stability,based on which the procedure for activation was optimized and verified.Results Using polysaccharide derivation rate as an indicator,pH value and amount of activating agent added showed significant effect on activation of group Y meningococcal polysaccharide(P < 0.05).However,using free polysaccharide content as an indicator,the effects of three factors on activation of the polysaccharide could not be evaluated(P > 0.05).Based on the determination results of various indexes and the stability of polysaccharide-TT conjugate,the procedure for activation of polysaccharide was optimized as follows:each microgram of polysaccharide was added with 1 mg of activating agent,and reacted at pH12 for 10 min.Three batches of group Y meningococcal polysaccharide-TT conjugate were prepared by the optimized procedure,of which all the quality indexes met the routine quality control standard for polysaccharide conjugate vaccine.Conclusion The group Y meningococcal polysaccharide-protein conjugate vaccine with low free polysaccharide content and high immunogenicity was prepared by the optimized polysaccharide activation procedure which showed high reproducibility.更多还原