正交试验法优化b型流感嗜血杆菌多糖活化工艺

Optimization of polysaccharide activation process of Haemophilus influenzae type b by orthogonal test

目的优化b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)多糖活化工艺,提高Hib结合疫苗的产量和质量。方法设计三因素三水平正交试验,考察不同pH、溴化氰加量和活化时间对Hib多糖衍生物、多糖蛋白结合物的影响,依据正交试验结果确定最优的多糖活化工艺,并对该工艺进行放大和验证。结果多糖活化时pH和活化时间对多糖衍生物衍生率及重均分子量(M_(w))均影响显著(P均<0.05),溴化氰加量对多糖衍生物衍生率影响差异有统计学意义(P<0.05),但对衍生物M的影响差异无统计学意义(P>0.05)。多糖衍生物衍生率和M与多糖蛋白结合物的多糖回收率、多糖与蛋白比值、高分子结合物含量、K_(D)≤0.2的多糖回收率、GMT各指标均显著相关(P均<0.05)。综合多糖蛋白结合物产量、免疫原性及稳定性。确定的最优多糖活化工艺为:在pH 10.5条件下,加入与精制多糖等重的溴化氰反应5 min。结论经优化的多糖活化工艺可以用于Hib多糖蛋白结合物规模化的生产。

Objective To optimize the polysaccharide activation process of Haemophilus influenzae type b(Hib)and improve the production and quality of Hib conjugate vaccine.Methods A three-factor three-level orthogonal test was designed to study the effects of different pH,cyanogen bromide addition and activation time on the Hib polysaccharide derivatives and polysaccharide-protein conjugates.The optimal activation process of capsular polysaccharide was determined based on the test results and was scaled up and validated.Results The effects of pH and activation time during polysaccharide activation on the derivatization rate and heavy average molecular weight(M_(w))of polysaccharide derivatives were significant(both P<0.05);and the difference of cyanogen bromide addition on the derivatization rate of polysaccharide derivatives was statistically significant(P<0.05);but the difference on the derivative Mwas not statistically significant(P>0.05).The polysaccharide derivative derivative rate and Mwere significantly correlated with the each index of polysaccharide recovery of polysaccharide protein conjugates,polysaccharide to protein ratio,polymer conjugate content,polysaccharide recovery for K_(D)≤0.2,and GMT(all P<0.05).The optimal polysaccharide activation process determined in combination by combining the polysaccharide protein conjugate yield,immunogenicity and stability:the reaction was carried out by adding cyanogen bromide of equal weight with refined polysaccharide for 5 min at pH 10.5.Conclusion The optimized polysaccharide activation process can be used for the production of Hib polysaccharide protein conjugates on a large scale.