气相色谱法对狂犬病疫苗灭活工艺中β-丙内酯研究

doi:10.13523/j.cb.20190604

中国生物工程杂志

2019, Vol. 39

Issue (6): 25-31 DOI: 10.13523/j.cb.20190604

研究报告

气相色谱法对狂犬病疫苗灭活工艺中β-丙内酯研究

高彦,杜晶晶,王斌,刘琦,申志强(

)

华兰生物工程股份有限公司研发中心新乡 453000

Study on β-Propiolactone in Inactivation Process of Rabies Vaccine by Gas Chromatography

Yan GAO,Jing-jing DU,Bin WANG,Qi LIU,Zhi-qiang SHEN(

)

Hualan Biological Engineering, INC R&D Center, Xinxiang 453000, China

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摘要：

目的: 基于GC-FID法,对狂犬病疫苗生产过程中采用的β-丙内酯灭活剂进行了含量及稳定性研究。方法: 气相色谱条件,采用Agilent DB-624(30m×0.530mm×3.00μm)毛细管柱;升温程序,初始温度为80℃,保持1min,以20℃/min的速率升温至200℃,保持3min;色谱柱流量,3ml/min;检测器温度,250℃;进样口温度,150℃;载气,氮气,线速度,25cm/s;进样量,1μl,分流比为2∶1;进样方式,手动进样。结果: 以乙腈作为稀释剂,BPL在1∶100~1∶32 000范围内线性关系良好(R ²≥0.999)。在1∶200、1∶1 000、1∶8 000 三个浓度水平下,加标回收率在95.04%~116.86%,相对标准偏差(RSD)为2.6%~3.2%,检测限为0.112μg/ml。结论: 方法简便、专属性强、稳定且在室温条件下操作,大大降低了对试验条件和技术操作的要求,能够满足灭活狂犬病病毒工艺中对BPL检测的需求。

关键词： 狂犬病疫苗; β-丙内酯; 气相色谱

Abstract:

Objective: According to GC-FID method, the content and stability of β-propiolactone which was used as an inactivator in the preparation of rabies vaccines were systematically studied. Methods: Gas chromatography condition: Agilent DB-624 (30m×0.530mm×3.00μm)capillary column was adopted. The temperature program: Initial temperature of 80℃ was maintained for 1min, then was raised to 200℃ at the rate of 20℃/min and was maintained for 3min. Column flow: 3ml/min. The temperatures of detector and inlet were 250℃ and 150℃ respectively. The linear velocity of the carrier gas (nitrogen) was set to 25cm/s. The injection volume was 1μl and the split ratio was 2∶1. The sampling mode: Manual injection. Results: BPL showed good linear relationship in the range of 1∶100-1∶32 000 with a correlation coefficient more than 0.999. A recovery study of BPL at three different concentration (1∶200, 1∶1 000, 1∶8 000) of spiked samples with range from 95.04% to 116.86% with RSD less than 3.2% further demonstrated the reliability and feasibility of this method. Moreover, the LOD of established method can be achieved as low as 0.112μg/ml. Conclusion The method is simple, specific, stable and can be carried out at room temperature with a satisfactory result, which reduces the requirements of complicated operation. In a word, the proposed method can meet the need that determines BPL in the process of inactivating rabies virus.

Key words: Rabies vaccine β-Propiolactone(BPL) Gas chromatography

收稿日期: 2018-10-25 出版日期: 2019-07-12

ZTFLH:

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通讯作者: 申志强 E-mail: szq3219@hualan.com

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引用本文:

高彦,杜晶晶,王斌,刘琦,申志强. 气相色谱法对狂犬病疫苗灭活工艺中β-丙内酯研究[J]. 中国生物工程杂志, 2019, 39(6): 25-31.

Yan GAO,Jing-jing DU,Bin WANG,Qi LIU,Zhi-qiang SHEN. Study on β-Propiolactone in Inactivation Process of Rabies Vaccine by Gas Chromatography. China Biotechnology, 2019, 39(6): 25-31.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190604 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I6/25

图1 β-丙内酯水解图

图2 不同乙腈比例下的BPL回收率

图3 各溶液对应的气相色谱图

图4 BPL峰面积-质量浓度的标准曲线图

表1 BPL在不同浓度梯度下的加标回收率

表2 气相色谱法检测BPL精密度结果分析表

图5 室温条件下BPL响应变化趋势图

图6 2~8℃时BPL在不同体系下降解速率考察

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