气相色谱法对狂犬病疫苗灭活工艺中β-丙内酯研究
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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(R2≥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。
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( 30 m × 0. 530 mm × 3. 00μm) capillary column was adopted. The temperature program: Initial temperature of 80℃ was maintained for 1 min,then was raised to 200℃ at the rate of 20℃/min and was maintained for 3 min. Column flow: 3 ml/min. The temperatures of detector and inlet were 250℃ and 150℃ respectively. The linear velocity of the carrier gas( nitrogen) was set to 25 cm/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.
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( 30 m × 0. 530 mm × 3. 00μm) capillary column was adopted. The temperature program: Initial temperature of 80℃ was maintained for 1 min,then was raised to 200℃ at the rate of 20℃/min and was maintained for 3 min. Column flow: 3 ml/min. The temperatures of detector and inlet were 250℃ and 150℃ respectively. The linear velocity of the carrier gas( nitrogen) was set to 25 cm/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.
引文
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[5]国家药品监督管理局.生物制品生产工艺过程变更管理技术指导原则.[2018-09-15]. http://www. sda. gov. cn/WS01/CL0844/9350. html.State Drug Administration. Biological product production process change management techn-ology guiding principles.[2018-09-15]. http://www. sda. gov. cn/WS01/CL0844/9350. html.
[6]宋清爽,吴恩应,张运佳,等.血液制品病毒灭活及去除工艺进展.生物技术通讯,2012,23(4):627-630.Song Q S, Wu E Y, Zhang Y J, et al. Advance in virus inactivation and removal processes of blood products. Letters In Biotechnology,2012,23(4):627-630.
[7] She Y M,Cheng K D,Aaron Farnsworth,et al. Surface modifications of influenza proteins upon virus inactivation byβ-propiolactone. Proteomics,2013,13(23):3537-3547.
[8] Sasaki Y,Yoshino N,Sato S,et al. Analysis of the betapropiolactone sensitivity and optimization of inactivation methods for human influenza H3N2 virus. Journal of Virological Methods,2016,235(4):105-111.
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[11]杨健,高军,张庆义,等.β-丙内酯含量气相色谱检测方法的建立及其水解情况分析.中国生物制品学杂志,2010,23(3):323-324.Yang J, Gao J, Zhang Q Y, et al. Development of gas chromatography for determination of Propiolactone(BPL)content and analysis of BPL hydrolysis. Chinese Journal of Biologicals,2010,23(3):323-324.
[12]梁婧,李举,马继华,等.气相色谱法检测灭活狂犬病病毒浓缩液中β-丙内酯的含量.中国生物制品学杂志,2017,30(12):1317-1320.Liang J,Li J,Ma J H,et al. Determination ofβ-propiolactone content in inactivated rabies virus concentrate by gas chromatography. Chinese Journal of Biologicals,2017,30(12):1317-1320.
[13] Shan G Z,Ma X,Ji H,et al. Determination of residualβ-propiolactone in inactivated rabies virus concentrate by gas chromatography. China Pharmacy,2014,25(25):2357-2359.
[14]Xu Y,Wei R,Jiang Y,et al. Study on the determination ofβ-propiolactone in rotavirus vaccine by gas chromatography.Progress in Microbiology and Immunology,2010,38(4):21-23.
[15] Lei S, Gao X, Sun Y, et al. Gas chromatography-mass spectrometry method for determination ofβ-propiolactone in human inactivated rabies vaccine and its hydrolysis analysis.Journal of Pharmaceutical Analysis,2018,6(3):373-377.
[16]刘兆文,李福安,钱浩.β-丙内酯在人用纯化地鼠肾细胞狂犬疫苗制备中的应用研究.中国人兽共患病杂志,1999,15(6):71-72.Liu Z W, Li F A, Qian H. Study for application ofβ-propiolactone on preparation of human rabies vaccine of purified humster kidney cell. Chinese Journal of Zoonoses,1999,15(6):71-72.
[17]李福安,窦志勇,潘丽静.狂犬疫苗灭活后β-丙内酯水解的最佳pH值的选择.中国公共卫生,1999,15(11):964.Li F A,Dou Z Y,Pan L J. Selection of optimal pH for hydrolysis ofβ-propiolactone after rabies vaccine inactivation. Chinese Journal of Public Health Management,1999,15(11):964.
[18]徐守振,王新,尹燕博.四种不同灭活剂对新城疫病毒的灭活效果研究.中国家禽,2011,33(14):15-19.Xu S Z,Wang X,Yin Y B. Inactivation efficacy of newcastle disease virus with four different inactivants. China Poultry,2011,33(14):15-19.
[19]Delrue I,Verzele D,Madder A,et al. Inactivated virus vaccines from chemistry to prophylaxis:merits, risks and challenges.Expert Rev Vaccines,2012,11(6):695-719.
[20]龚文波,郝伟伟,廖韦萍,等.两种不同灭活剂对基因Ⅶ型新城疫病毒灭活效果的影响研究.四川畜牧兽医,2017,44(10):21-23.Gong W B,Hao W W,Liao W P,et al. Inactivation effect of genotypeⅦnewcastle disease virus with two different inactivants.Sichuan Animal and Veterinary Sciences,2017,44(10):21-23.
[21]冯若飞,樊得英,韦鹏建,等.β-丙内酯对脑心肌炎病毒灭活效果的试验.中国兽医杂志,2011,47(8):19-21.Feng R F, Fan D Y, Wei P J, et al. Experiment on the inactivation effect ofβ-propiolactone on encephalomyocarditis virus. Chinese Journal of Veterinary Medicine,2011,47(8):19-21.
[22]姜立民,林晓波,高磊,等.β-丙内酯对狂犬病病毒的灭活效果.国际流行病学传染病学杂志,2014,41(2):137-139.Jing L M,Lin X B,Gao L,et al. Inactivation of rabies virus withβ-propiolactone. Journal of the Chinese Medical Association,2014,41(2):137-139.
[23]国家药典委员会.中国药典.第三部.北京:中国医药科技出版社,2015:146-149.Chinese Pharmacopoeia Commission. Chinese pharmacopoeia.Third part. Beijing:China Medical Science and Technology Press,2015:146-149.
[2]李义.狂犬病的诊断与防治研究.上海畜牧兽医通讯,2018,23(4):56-57.Li Y. Diagnosis and prevention of rabies. Shanghai Livestock and Veterinary Communication,2018,23(4):56-57.
[3]蔡黎,朱政纲,文丽,等.人用狂犬病疫苗研究进展.药物流行病学杂志,2017,26(12):841-844.Cai L,Zhu Z G,Wen L,et al. Progress in research on human rabies vaccine. Chinese Journal of Pharmacoepidemiology,2017,26(12):841-844.
[4]王春雨,徐琳凯,李超,等.工艺处理过程中病毒的灭活效果研究.实验动物科学,2015,32(2):15-17.Wang C Y,Xu L K,Li C,et al. Study on the inactivation effect of virus during process. Laboratory Animal Science,2015,32(2):15-17.
[5]国家药品监督管理局.生物制品生产工艺过程变更管理技术指导原则.[2018-09-15]. http://www. sda. gov. cn/WS01/CL0844/9350. html.State Drug Administration. Biological product production process change management techn-ology guiding principles.[2018-09-15]. http://www. sda. gov. cn/WS01/CL0844/9350. html.
[6]宋清爽,吴恩应,张运佳,等.血液制品病毒灭活及去除工艺进展.生物技术通讯,2012,23(4):627-630.Song Q S, Wu E Y, Zhang Y J, et al. Advance in virus inactivation and removal processes of blood products. Letters In Biotechnology,2012,23(4):627-630.
[7] She Y M,Cheng K D,Aaron Farnsworth,et al. Surface modifications of influenza proteins upon virus inactivation byβ-propiolactone. Proteomics,2013,13(23):3537-3547.
[8] Sasaki Y,Yoshino N,Sato S,et al. Analysis of the betapropiolactone sensitivity and optimization of inactivation methods for human influenza H3N2 virus. Journal of Virological Methods,2016,235(4):105-111.
[9]Chen W,Li Z,Liu J,et al. Application ofβ-propiolactone in HFRS vaccine. Chinese Journal of Public Health,2003,19(6):669-670.
[10] Zhang Y H,Gao C, Pan G,et al. Determination ofβ-propiolactone residues in human rabies vaccine for Vero cells.Chinese Journal of Biologicals,1998,11(3):144.
[11]杨健,高军,张庆义,等.β-丙内酯含量气相色谱检测方法的建立及其水解情况分析.中国生物制品学杂志,2010,23(3):323-324.Yang J, Gao J, Zhang Q Y, et al. Development of gas chromatography for determination of Propiolactone(BPL)content and analysis of BPL hydrolysis. Chinese Journal of Biologicals,2010,23(3):323-324.
[12]梁婧,李举,马继华,等.气相色谱法检测灭活狂犬病病毒浓缩液中β-丙内酯的含量.中国生物制品学杂志,2017,30(12):1317-1320.Liang J,Li J,Ma J H,et al. Determination ofβ-propiolactone content in inactivated rabies virus concentrate by gas chromatography. Chinese Journal of Biologicals,2017,30(12):1317-1320.
[13] Shan G Z,Ma X,Ji H,et al. Determination of residualβ-propiolactone in inactivated rabies virus concentrate by gas chromatography. China Pharmacy,2014,25(25):2357-2359.
[14]Xu Y,Wei R,Jiang Y,et al. Study on the determination ofβ-propiolactone in rotavirus vaccine by gas chromatography.Progress in Microbiology and Immunology,2010,38(4):21-23.
[15] Lei S, Gao X, Sun Y, et al. Gas chromatography-mass spectrometry method for determination ofβ-propiolactone in human inactivated rabies vaccine and its hydrolysis analysis.Journal of Pharmaceutical Analysis,2018,6(3):373-377.
[16]刘兆文,李福安,钱浩.β-丙内酯在人用纯化地鼠肾细胞狂犬疫苗制备中的应用研究.中国人兽共患病杂志,1999,15(6):71-72.Liu Z W, Li F A, Qian H. Study for application ofβ-propiolactone on preparation of human rabies vaccine of purified humster kidney cell. Chinese Journal of Zoonoses,1999,15(6):71-72.
[17]李福安,窦志勇,潘丽静.狂犬疫苗灭活后β-丙内酯水解的最佳pH值的选择.中国公共卫生,1999,15(11):964.Li F A,Dou Z Y,Pan L J. Selection of optimal pH for hydrolysis ofβ-propiolactone after rabies vaccine inactivation. Chinese Journal of Public Health Management,1999,15(11):964.
[18]徐守振,王新,尹燕博.四种不同灭活剂对新城疫病毒的灭活效果研究.中国家禽,2011,33(14):15-19.Xu S Z,Wang X,Yin Y B. Inactivation efficacy of newcastle disease virus with four different inactivants. China Poultry,2011,33(14):15-19.
[19]Delrue I,Verzele D,Madder A,et al. Inactivated virus vaccines from chemistry to prophylaxis:merits, risks and challenges.Expert Rev Vaccines,2012,11(6):695-719.
[20]龚文波,郝伟伟,廖韦萍,等.两种不同灭活剂对基因Ⅶ型新城疫病毒灭活效果的影响研究.四川畜牧兽医,2017,44(10):21-23.Gong W B,Hao W W,Liao W P,et al. Inactivation effect of genotypeⅦnewcastle disease virus with two different inactivants.Sichuan Animal and Veterinary Sciences,2017,44(10):21-23.
[21]冯若飞,樊得英,韦鹏建,等.β-丙内酯对脑心肌炎病毒灭活效果的试验.中国兽医杂志,2011,47(8):19-21.Feng R F, Fan D Y, Wei P J, et al. Experiment on the inactivation effect ofβ-propiolactone on encephalomyocarditis virus. Chinese Journal of Veterinary Medicine,2011,47(8):19-21.
[22]姜立民,林晓波,高磊,等.β-丙内酯对狂犬病病毒的灭活效果.国际流行病学传染病学杂志,2014,41(2):137-139.Jing L M,Lin X B,Gao L,et al. Inactivation of rabies virus withβ-propiolactone. Journal of the Chinese Medical Association,2014,41(2):137-139.
[23]国家药典委员会.中国药典.第三部.北京:中国医药科技出版社,2015:146-149.Chinese Pharmacopoeia Commission. Chinese pharmacopoeia.Third part. Beijing:China Medical Science and Technology Press,2015:146-149.