无细胞百白破联合疫苗腺苷酸环化酶毒素液质联用定量检测方法的建立和应用  

The establishment and application of a quantitative LC-MS/MS method for detection of adenylate cyclase toxin in DTaP combined vaccine

作　　者：卫辰[1] 吴燕[1] 蔡心怡 晁哲[1] 王丽婵 马霄[1] WEI Chen;WU Yan;CAI Xin-yi;CHAO Zhe;WANG Li-chan;MA Xiao(National Institutes for Food and Drug Control,Key laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京102629 [2]武汉生物制品研究所有限责任公司细菌类疫苗室,湖北武汉430200

出　　处：《微生物学免疫学进展》2022年第1期22-29,共8页Progress In Microbiology and Immunology

基　　金：国家“重大新药创制”科技重大专项(2018ZX097380052017)。

摘　　要：目的 建立不同工艺配方的无细胞百白破联合疫苗(diphtheria, tetanus and acellular Pertussis combined vaccine, DTaP)中腺苷酸环化酶毒素(adenylate cyclase toxin, ACT)的液质联用(liquid chromatography-tandem mass spectrometry, LC-MS/MS)定量检测方法。方法 对百日咳疫苗精制液、原液、DTaP成品和ACT参考品进行酶解前处理,采用BioC18 (2.1 mm×150 mm, 3μm)为固定相,0.1%甲酸水溶液和0.1%甲酸乙腈溶液为流动相,梯度洗脱,流速为0.3 mL/min,柱温为35℃,多反应监测模式(multiple reaction monitoring, MRM)对特定离子对进行检测。以ACT参考品建立标准曲线,对样品中的ACT准确定量,并对方法进行验证及初步应用。结果 肽段ITGDAQANVLR特异性良好,在1.25~31.25μg/mL范围内线性良好(R^(2)>0.99),残差平方和为2.55,不同基质稀释标准曲线的残差平方和分别为3.08和1.32,基质效应较弱。DTaP成品回收率为80%~109%、原液回收率为93%~121%、精制液回收率为112%~120%;进样精密度为2.32%;中间精密度<20%,具备较好的准确性、精密度,且不同型号仪器的系统适用性研究结果一致。将该方法用于检测4个厂家的无细胞百白破联合疫苗多个工序段含有百日咳疫苗的样品,其中2个厂家的样品均有ACT检出,且ACT在共纯化阶段不断富集达100~250倍,另2个厂家产品仅在发酵液和盐析液中检出。结论 建立的LC-MS/MS检测方法准确性和精密度都比较好,基质效应弱,可用于DTaP中ACT的定量检测和工艺过程监测。Objective To establish a quantitative LC-MS/MS method to detect the content of ACT in DTaP produced by different processes and formulations. Methods The pertussis refined solution, pertussis bulk, DTaP final product and ACT reference were pretreated by enzymatic hydrolysis. Use Bio C18(2.1 mm×150 mm, 3 μm) as the stationary phase, and 0.1% formic acid in water-0.1% formic acid in acetonitrile as the mobile phase, the gradient elution was conducted at a flow rate of 0.3 mL/min, and the column temperature was 35 ℃. Specific ion-pair was detected by MRM mode. A standard curve was established using the ACT reference, and the ACT in the sample was accurately quantified. This method was validated and initially applied. Results Peptide ITGDAQANVLR had good specificity and linearity in the range of 1.25-31.25 μg/mL(R^(2)>0.99) and the residual sum of squares(SSR) was 2.55. The SSRs of the standard curves of different matrix dilutions were 3.08 and 1.32, respectively, and the matrix effect was weak. The recovery rates of DTaP final products, bulks and refined solutions were 80%~109%, 93%~121%, and 112%~120%, respectively. The sample injection precision was 2.32% and the intermediate precision was < 20%, which showed that this method had good accuracy and precision. Furthermore, this method had good adaptability in instrument systems from different manufacturers. This method was used to detect samples from multiple process stages of DTaP vaccine production from four manufacturers. Among them, ACT were detected in all samples from two manufacturers, and was continuously enriched by 100~250 times during the co-purification stage. ACT was only detected in the samples of fermentation broth and salting-out liquid from the other two manufacturers. Conclusion The LC-MS/MS detection method had good accuracy, precision and weak matrix effect, which can be used for quantitative detection of ACT in DTaP and process monitoring.

关 键 词：无细胞百白破联合疫苗 腺苷酸环化酶毒素 质量控制 液相色谱串联质谱 

分 类 号：Q656.31[生物学—生物物理学]