陶瓷羟基磷灰石层析法纯化b型流感嗜血杆菌荚膜多糖

Purification of capsular polysaccharide of Haemophilus influenzae type b by ceramic hydroxyapatite chromatography

目的 采用陶瓷羟基磷灰石(ceramic hydroxyapatite,CHT)层析法纯化b型流感嗜血杆菌荚膜多糖,以代替传统的苯酚抽提法。方法 将b型流感嗜血杆菌粗多糖溶解后,采用4种方案进行粗多糖纯化,筛选最佳工艺路线;并对工艺路线进行优化(乙醇沉淀条件、切向流超滤条件、CHT层析条件)。结果 根据工艺路线选择及优化结果,确定如下工艺路线:将b型流感嗜血杆菌粗多糖溶解后经35%和80%浓度乙醇沉淀2次,沉淀物溶解后,经切向流超滤10次,CHT层析收获流穿液,再经切向流超滤后冻干。最终获得的b型流感嗜血杆菌多糖质量符合《中国药典》三部(2015版)要求。结论 可使用CHT层析法纯化b型流感嗜血杆菌荚膜多糖。相比于传统苯酚抽提工艺,该方法环保、安全。

Objective To purify the capsular polysaccharide of Haemophilus influenzae type b by ceramic hydroxyapatite chromatography(CHT) instead of traditional phenol extraction. Methods The crude polysaccharide of Haemophilus influenzae type b was dissolved and purified by four protocols to screen the purification procedure. The screened procedure was optimized,including the conditions for ethanol precipitation,tangential flow ultrafiltration and CHT.Results According to the results of screening and optimization,the purification procedure was determined as follows:the crude polysaccharide of Haemophilus influenzae type b was dissolved and precipitated with 35% and 80% ethanol respectively;the precipitate was dissolved and subjected to tangential flow ultrafiltration for 10 times;the flow through fluid was harvested by CHT,subjected to tangential flow ultrafiltration and lyophilized. The quality of obtained polysaccharide met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Conclusion The capsular polysaccharide of Haemophilus influenzae type b may be purified by CHT. Compared with the traditional phenol extraction process,the method is environment-friendly and safe.