层析法纯化C群脑膜炎球菌多糖及其制备的多糖蛋白结合物的安全性和免疫原性  

Purification of group C meningococcal capsular polysaccharide by chromatography as well as safety and immunogenicity of prepared polysaccharide-protein conjugate

作　　者：王雪薇[1,2] 刘钰[1,2] 陈敬[1,2] 焦小玲[1,2] 王平[1,2] 张燕斌[1,2] 林海涛[1,2] 刘梅影[1,2] 

机构地区：[1]北京微谷生物医药有限公司,北京101111 [2]北京生物制品研究所有限责任公司,北京101111

出　　处：《中国生物制品学杂志》2013年第12期1705-1710,1714,共7页Chinese Journal of Biologicals

基　　金：重大新药创制科技重大专项(2010ZX09401-303-3-08)

摘　　要：目的采用层析法纯化C群脑膜炎球菌多糖,以替代冷酚抽提法,并对制备的多糖蛋白结合物进行初步安全性和免疫原性评价。方法采用温和的表面活性剂去氧胆酸钠对C群脑膜炎球菌多糖进行前处理,并用Capto adhere和Capto DEAE阴离子交换凝胶柱串联,对多糖进行柱层析纯化,再通过脱盐去除残留的去氧胆酸钠,获得精制多糖。对缓冲液中的去氧胆酸钠浓度、缓冲液pH值、样品的前处理时间及离子交换流速进行优化,并对建立的层析纯化工艺进行放大,纯化3批多糖,按照《中国药典》三部(2010版)要求检测各项质控指标,并通过核磁共振-氢谱检测纯化后的多糖结构。将层析法和苯酚法纯化的多糖分别与破伤风类毒素结合,制备多糖蛋白结合物,对结合物进行异常毒性、小鼠急性毒性试验和免疫原性检测。结果经优化,确定缓冲液的去氧胆酸钠浓度为1.0%,pH值为8.0,样品前处理时间为6 h,离子交换流速为2.0 ml/min。放大工艺制备的3批C群脑膜炎球菌多糖各项检测指标均合格,多糖回收率均大于70%,明显高于苯酚法制备的多糖。核磁共振-氢谱显示,层析法与苯酚法纯化的多糖主要峰基本一致,层析法未改变多糖结构。层析法纯化的多糖制备的多糖蛋白结合物异常毒性试验合格,小鼠急性毒性试验结果与苯酚法纯化多糖制备的多糖蛋白结合物比较,差异无统计学意义(P>0.05),免疫小鼠后均具有良好的免疫原性。结论层析法分离杂蛋白操作简便,省时省力,工艺易于放大,且减少了对环境的污染,可替代苯酚抽提法用于C群脑膜炎球菌多糖的纯化。Objective To purify group C meningococcal capsular polysaccharide by chromatography instead of cold phenol extraction and preliminarily evaluate the safety and immunogenicity of polysaccharide-protein conjugate prepared. Methods Group C meningococcal capsular polysaccharide was pre-treated with mild surfactant sodium deoxycholate and purified by anion-exchange chromatography with Capto adhere and Capto DEAE columns, from which residual sodium deoxycholate was removed by desahing. The sodium deoxycholate concentration, pH value of buffer, time for pretreatment and flow rate for anion exchange were optimized, and the developed procedure was scaled up. The batches of group C meningococcal capsular polysaccharides were purified by the developed procedure, then subjected to overall control tests according to the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2010 edition）, and analyzed for structure by ^1H-nuclear magnetic resonance （NMR）. Polysaceharide-tetanus toxoid （TT） conjugates were prepared by conjugating the polysaccharide purified by chromatography and cold phenol extraction to TT respectively, and tested for abnormal toxicity, acute toxicity in mice and immunogenicity. Results The optimal sodium deoxycholate concentration, pH value of buffer, time for pre-treatment and flow rate for anion exchange were 1. 0%, 8. 0, 6 h and 2. 0 ml / min. Three batches of polysaccharides purified by the developed procedure were qualified in overall control tests, while the recovery ofpolysaccharide was more than 70%, which was significantly higher than that purified by cold phenol extraction. The ~H-NMR spectra showed that the main peaks of polysaccharides purified by chromatography and cold phenol extraction were basically in agreement, indicating no change of structure of polysaccharide purified by chromatography. The conjugate prepared with polysaccharide purified by chromatography was qualified in abnormal toxicity test and showed good immunogenicity in mice, of which the acute toxicity test result showed

关 键 词：C群脑膜炎球菌 多糖 阴离子交换层析 结合物 安全性 免疫原性 

分 类 号：R378.15[医药卫生—病原生物学] O658.1[医药卫生—基础医学]