C、Y、W 135群脑膜炎球菌荚膜多糖化学结构核磁共振检测方法的建立

Development of nuclear magnetic resonance method for analysis of chemical structure of groups C, Y and W135 Neisseria meningococcal capsular polysaccharides

目的建立基于核磁共振(nuclear magnetic resonance,NMR)技术检测C群脑膜炎球菌荚膜多糖(group C meningococcal capsular polysaccharide,GCMP)、Y群脑膜炎球菌荚膜多糖(GYMP)和W135群脑膜炎球菌荚膜多糖(GWMP)化学结构的方法。方法建立1H-NMR检测方法:使用布鲁克600 MHz超导傅里叶核磁共振谱仪,配置5 mm BBO正向宽带高分辨液体探头。脉冲程序为水峰压制(zgpr),温度为298 K(25℃)。具体参数设置如下:中心频率约为4.7 ppm;图谱宽度为7 211 Hz;采样时间为2.272 s;弛豫延迟时间为2 s;扫描次数为512次;信号经傅里叶转换后加入1.0 Hz的窗口函数,检测结果使用Topspin 3.0软件进行处理。验证该方法的专属性、重复性及中间精密性。结果 5批GCMP、GYMP和GWMP检测结果的CV值均小于0.35%;3个浓度GCMP、GYMP和GWMP在相同的操作条件下以及不同工作日间、不同实验员间的检测结果的CV值均小于0.35%。结论建立的1H-NMR检测方法简便、快速、准确,检测GCMP、GYMP和GWMP具有极高的专属性、重复性和中间精密性,适用于疫苗生产研究中GCMP、GYMP和GWMP样品的定性结构鉴定。

Objective To develop a nuclear magnetic resonance （NMR） method for analysis of chemical structures of Neisseria meningoeoceal capsular polysaccharides of groups C, Y and W135, shortened as GCMP, GYMP and GWMP, respectively. Methods The IH-NMR method was developed by using Bruker AVANCE III 600 MHz Superconducting Fourier NMR Spectrometer with a 5 mm positive broadband high-resolution liquid probe. The pulse program was zgpr （water suppression experiment）. The IH-NMR spectra were collected at 298 K （25 ℃） with a spectral width of 7211 Hz, a cquisition time of 2. 272 s and a relaxation delay of 2 s, accumulating 512 scans. The spectra were weighted with 1.0 Hz line broadening and Fourier transformed. For data acquisition and processing, Topspin version 3. 0 software was used. The method was verified for specificity, repeatability and intermediate precision. Results The CVs of determination results of each five batches of GCMP, GYMP and GWMP were less than 0. 35%, However, CVs of determination results of GCMP, GYMP and GWMP at three concentrations under the same operating condition on various working dates by various personnel were less than 0. 35%. Conclusion The developed IH-NMR method was simple, rapid and accurate, which showed high specificity, repeatability and intermediate precision in determination of GCMP, GYMP and GWMP, and was suitable for the structural analysis of the three groups of Neisseria meningococcal capsular polysaccharides in research andproduction of vaccine.