4型肺炎球菌多糖丙酮酸含量核磁共振测定方法的建立及验证  

Development and validation of a nuclear magnetic resonance method for determination of pyruvic acid in pneumococcal serotype 4 polysaccharide

作　　者：杜娇 周海飞 李献林 魏开琦 马凯凯 张钰奇 刘爱萍 李阿妮 罗树权 DU Jiao;ZHOU Hai-fei;LI Xian-lin;WEI Kai-qi;MA Kai-kai;ZHANG Yu-qi;LIU Ai-ping;Li A-ni;LUO Shu-quan(The First Research Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司第一研究室、甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2023年第2期18-24,共7页Progress In Microbiology and Immunology

基　　金：国家科技重大专项重大新药创制(2018ZX09738-006)。

摘　　要：目的 建立检测4型肺炎球菌多糖侧链上的丙酮酸(pyruvic acid, PA)含量的定量核磁共振(quantitativenuclear magnetic resonance, qNMR)方法,并进行验证。方法 用以3-(三甲基硅)丙酸-D4钠盐为内标的重水作为溶剂,在600 MHz核磁共振仪上采集波谱,采集参数为:脉冲程序zg30,弛豫延迟时间9 s,扫描次数512次。对方法进行线性、范围、专属性、准确度、精密度、耐用性验证。结果 PA含量在0.10~12.00 mg/mL范围内线性良好,且R~2> 0.99。以PA上的甲基质子峰(化学位移1.54 ppm)为特征峰测定PA信号峰,qNMR检测方法专属性良好。两种方法的回收率分别在93.52%~101.37%和92.71%~100.10%之间;建立的方法重复性及中间精密度良好,不同PA含量测定值RSD分别为3.67%,2.12%和3.70%;改变温度和扫描次数,2种条件的测定结果差异均无统计学意义(P>0.05)。结论 建立的4型肺炎球菌多糖中PA含量qNMR检测方法简便、切实有效,为13价肺炎球菌结合疫苗的生产提供了一种可靠的产品放行或过程监测方法。Objective Quantitative nuclear magnetic resonance(qNMR) method was established and validated to determine the content of pyruvic acid(PA) in the side chain of pneumococcal serotype 4 polysaccharide. Methods Spectra was obtained in deuterium oxide using trimethylsilyl propionate as the internal standard, and performed using 600 MHz spectrometer with the following acquisition parameters: zg30-pulse, relaxation delay-9 s, number of scans-512. Validation of the quantitative method was also performed in terms of linearity, range, specificity, accuracy, precision and ruggedness. Results The content of PA has an excellent linearity in the range of 0.10-12.00 mg/mL, and the regression coefficient(R~2 value) was above 0.99;the signal was determined taking methyl proton on PA at δ1.54 ppm as the characteristic peak, and the qNMR method showed good specificity. The recovery rates of two methods were between 93.52%-101.37% and 92.71%-100.10%, respectively. The method has good repeatability and intermediate precision, the RSDs of PA determination results were 3.67%, 2.12% and 3.70%, respectively. The determination results showed no significant difference(P0.05) after adjusting the temperature and the number of scans. Conclusion The developed qNMR method for the detection of pyruvic acid content in pneumococcal serotype 4 polysaccharide was a simple and practical method, which can be used as a reliable product release or process monitoring method for the vaccine manufacturers as per regulatory requirements.

关 键 词：4型肺炎球菌 多糖 肺炎球菌多糖结合疫苗 丙酮酸 定量核磁共振 方法验证 

分 类 号：R392-33[医药卫生—免疫学]