层析法提纯A、C、Y、W135群脑膜炎球菌荚膜多糖工艺的建立

目的建立A、C、Y、W135群脑膜炎球菌荚膜多糖层析纯化工艺。方法采用陶瓷羟基磷灰石和DEAE Sepharose FF层析柱,在PBS体系中纯化A群多糖。以0.5%脱氧胆酸钠预处理C群多糖,用陶瓷羟基磷灰石层析柱在PBS体系中纯化C群多糖。以含有脱氧胆酸钠的平衡缓冲液溶解Y和W135群多糖,再用Capto Adhere和Capto DEAE层析柱串联纯化。纯化的多糖经Sephadex G-25 Medium层析柱脱盐后冻干,按中国药典2015年版三部的要求进行检定。结果经过层析纯化,A、C、Y、W135群多糖的蛋白质含量分别降至3.7、4.2、5.4和5.3 mg/g,核酸含量分别降至1.2、3.0、1.1和0.8 mg/g,均符合药典要求。此外,磷、唾液酸和O-乙酰基含量等指标亦均符合药典标准。结论建立了A、C、Y、W135群脑膜炎球菌荚膜多糖的层析纯化工艺。

Purification process development of capsular polysaccharides from Neisseria meningitidis serogroups A,C, Y,W135 by chromatography

Objective  To develop the chromatographic methods for purifying capsular polysaccharides from Neisseria meningitidis serogroups A, C, Y, W135. Methods  Group A capsular polysaccharide was purified by ceramic hydroxyapatite and DEAE sepharose FF chromatographic column in PBS system. Group C capsular polysaccharide was pre-treated with sodium deoxycholate (SDC), then purified by ceramic hydroxyapatite column in PBS system. Groups Y and W135 capsular polysaccharides were dissolved in equilibration buffer system containing SDC, then purified by Capto Adhere and Capto DEAE columns in upper and lower series. The purified polysaccharides were desalted with Sephadex G25 Medium column, freeze-dried, and subjected to control tests according to Chinese pharmacopoeia (Part Ⅲ of the 2015 edition). Results  After purification, the protein contents in groups A, C, Y, W135 polysaccharides decreased to 3.7, 4.2, 5.4 and 5.3 mg/g, respectively, and the nucleic acid contents decreased to 1.2, 3.0, 1.1 and 0.8 mg/g, respectively. All the above contents and phosphorus, sialic acid, and O-acetyl group contents met the pharmacopoeia requirements. Conclusion The chromatographic methods for purifying capsular polysaccharides from Neisseria meningitidis groups A, C, Y and W135 are well developed.