高效阴离子交换色谱-脉冲安培法检测冻干b型流感嗜血杆菌结合疫苗的游离多糖含量  

Determination of free polysaccharide content in freeze-dried haemophilus influenzae type b conjugate vaccines by high performance anion exchange chromatography-pulsed amperometric detector

作　　者：贾松华 邓海清[1] 杜闪 张艳红[1] 尹珊珊[1] 刘建凯[1] JIA Song-hua;DENG Hai-qing;DU Shan;ZHANG Yan-hong;YIN Shan-shan;LIU Jian-kai(R&D Center,Beijing Minhai Biotechnology Co.,Ltd.,Beijing 102600,China)

机构地区：[1]北京民海生物科技有限公司研发中心,102600

出　　处：《中国医药生物技术》2022年第6期518-526,共9页Chinese Medicinal Biotechnology

摘　　要：目的 建立高效阴离子交换色谱-脉冲安培(HPAEC-PAD)法检测冻干b型流感嗜血杆菌(Hib)结合疫苗中游离多糖含量的方法,并对其进行验证,为Hib结合疫苗的质量监控提供依据。方法 优化水解供试品的实验条件,利用HPAEC-PAD法检测供试品中多聚磷酸核糖基核糖醇(PRP)含量。色谱条件采用CarboPacPA10分析柱(2mm×250mm)与CarboPac PA10保护柱(2 mm×50 mm),氢氧化钠-醋酸钠溶液梯度洗脱,流速为0.25ml/min,脉冲安培检测器,进样体积为20μl。用乙醇分步沉淀法、C4固相萃取法和超速离心法分离Hib游离多糖,并对三种方法得到的结果进行比较。同时验证方法的系统适用性、特异性、线性、精密度及准确性。结果 确定水解条件为终浓度0.4mol/L的NaOH,于37℃振荡1.5 h。本公司生产的冻干Hib结合疫苗不可用C4固相萃取柱分离游离多糖,确定了超速离心联合HPAEC-PAD法为冻干Hib结合疫苗游离多糖检测的最适方法。该方法专属性、分离度良好;PRP浓度在0.1962~19.616μg/ml范围内与峰面积呈良好的线性关系,R2>0.99。3批供试品分别测定6次,RSD分别为4.1%、2.3%、3.3%,不同时间测得的中间精密度RSD分别为8.3%、3.6%和9.1%。游离多糖的加标回收率分别为102.9%、94.3%、92.0%,RSD分别为3.9%、8.7%、5.3%。结论 初步建立了冻干Hib结合疫苗游离多糖含量测定的超速离心联合HPAEC-PAD法。该方法具有良好的准确性、精密度及稳定性,可用于冻干Hib结合疫苗中游离多糖含量的检测。Objective To develop and verify a high performance anion exchange chromatography-pulsed amperometric detector(HPAEC-PAD)method for determination of free polysaccharide content in freeze-dried Hib conjugate vaccines and provide a basis for quality control of freeze-dried Hib conjugate vaccines.Methods The condition for hydrolysis was optimized. The condition for HPAEC-PAD was as follows: CarboPac PA10 analytical column(2 mm × 250 mm)and CarboPac PA10 guard column(2 mm × 50 mm)was used. The mobile phase was a sodium hydroxide/sodium acetate with gradient elution at a flow rate of 0.25 ml/min. The samples were detected by pulsed amperometric detector. The injection volume was 20 μl. Meanwhile, the free polysaccharide of the same batch of vaccine were separated by ultracentrifugation or C4 solid phase extraction, and the results were compared with that by ethanol precipitation. The method was validated for system suitability, specificity, linearity, precision and accuracy.Results The condition for hydrolysis was optimized as treatment with 0.4 mol/L sodium hydroxide at 37 ℃ in oscillation for 1.5 h.C4 solid phase extraction wasn’t used as an alternative method of traditional ethanol precipitation method for free polysaccharide separation of the freeze-dried Hib conjugate vaccine produced by our company. And the best separative procedure of Hib free polysaccharide was determined. The method showed high specificity and separation degree in determination of free polysaccharide content in freeze-dried Hib conjugate vaccines. PRP at a concentration range of 0.1962-19.616 μg/ml showed good linear relationship to the peak area(R2 > 0.99). The RSDs of six test results of samples were 4.1%, 2.3% and 3.3%, while those determined at 3 time points were 8.3%, 3.6% and 9.1%, respectively. The recovery rates of free polysaccharide in the three batches of samples were 102.9%, 94.3% and 92.0%, with RSDs of 3.9%, 8.7% and 5.3%, respectively.Conclusion The ultracentrifugation combined with HPAEC-PAD method was preliminarily est

关 键 词：高效阴离子交换色谱-脉冲安培法 B型流感嗜血杆菌结合疫苗 超速离心 C4固相萃取 游离多糖 

分 类 号：R392[医药卫生—免疫学]