目的 优化b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)多糖纯化工艺,替代传统的苯酚结合乙醇方法.  方法 采用十六烷基三甲基溴化铵提取粗糖,以脱氧胆酸钠结合乙醇多级纯化进一步纯化粗糖,通过超滤浓缩和除菌过滤获得纯化的Hib多糖.采用优化的纯化工艺以中试和放大规模各纯化3批Hib多糖,按照《中华人民共和国药典》2010年版三部的要求检测纯化的Hib多糖.   结果 中试和放大规模纯化的各3批Hib多糖的相对分子质量分别为621 800、634 400、659 900和597 200、612 100、583 400,均符合规定的标准.中试和放大规模纯化的各3批Hib多糖的细菌内毒素含量分别为2.0、0.5、0.7 EU/μg和4.0、2.0、1.0 EU/μg,均明显低于规定的标准.鉴别试验显示,中试和放大规模纯化的各批Hib多糖均可与标准Hib抗血清形成明显的沉淀线.  结论 优化的Hib多糖纯化工艺具有较好的稳定性,且易于工艺放大,可替代传统工艺用于Hib多糖的纯化.

 Objective  To optimize the purification process of Haemophilus influenzae type b (Hib) polysaccharide for replacing the traditional combination of phenol and ethanol.  Methods   Crude Hib polysaccharides were extracted with Cetavlon and further purified with the combination of sodium deoxycholate and ethanol multistage purification, and purified polysaccharides were obtained by ultrafiltration and sterilizing filtration. Each 3 batches of purified Hib polysaccharides were prepared with the optimized purification precess in the pilot and amplifying scale. Prepared Hib polysaccharides were detected in accordance with the Chinese Pharmacopoeia 2010 edition (Volume Ⅲ).  Results   The relative molecular masses of each 3 batches of Hib polysaccharides purified in the pilot and amplifying scale were 621 800、634 400、659 900和597 200、612 100、583 400 respectively, and all met standard requirement. The contents of bacterial endotoxin of each 3 batch of Hib polysaccharides purified in the pilot and amplifying scale were 2.0, 0.5, 0.7 and 4.0, 2.0, 1.0 respectively, and were all significantly below standard requirement. The identity test showed each batch of Hib polysaccharides purified in the pilot and amplifying scale formed a strong precipitin line with antiserum to Hib.  Conclusion  The optimized purification process is stable and easy to scale-up, and can replace the traditional purification process of Hib polysaccharide.