高效液相体积排阻色谱法测定口蹄疫灭活疫苗146S 抗原含量及疫苗质量评估

doi:10.3864/j.issn.0578-1752.2019.20.019

中国农业科学 ›› 2019, Vol. 52 ›› Issue (20): 3695-3704.doi: 10.3864/j.issn.0578-1752.2019.20.019

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

高效液相体积排阻色谱法测定口蹄疫灭活疫苗146S 抗原含量及疫苗质量评估

朱元源¹,徐嫄¹,邹兴启¹,杨延丽²,刘丽丽²,万建青¹,李翠¹,徐璐¹,张乾义¹,夏应菊¹,王兆¹,郎洪武¹,王琴¹,张松平²,赵启祖¹()

¹ 中国兽医药品监察所，北京 100081
² 中国科学院过程工程研究所/生化工程国家重点实验室，北京 100190

收稿日期:2019-01-28 接受日期:2019-05-08 出版日期:2019-10-16 发布日期:2019-10-28
通讯作者: 赵启祖
作者简介:朱元源,E-mail：zhuyuanyzz@163.com。
基金资助:
国家重点研发计划资助(2018YFC1200501);国家重点研发计划资助(2016YFD0501500)

Determination of 146S Antigen in Inactivated Foot-and-Mouth Disease Vaccine by Size-Exclusion High-performance Liquid Chromatography and Quality Evaluation of Vaccine

YuanYuan ZHU¹,Yuan XU¹,XingQi ZOU¹,YanLi YANG²,LiLi LIU²,JianQing WAN¹,Cui LI¹,Lu XU¹,QianYi ZHANG¹,YingJu XIA¹,Zhao WANG¹,HongWu LANG¹,Qin WANG¹,SongPing ZHANG²,QiZu ZHAO¹()

¹ China Institute of Veterinary Drug Control, Beijing 100081
² National Key Laboratory of Biochemical Engineering/Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190

Received:2019-01-28 Accepted:2019-05-08 Online:2019-10-16 Published:2019-10-28
Contact: QiZu ZHAO

摘要/Abstract

摘要：

【背景】 口蹄疫疫苗质量评价方法对疫苗生产企业和监管部门进行质量控制尤为重要,146S抗原含量是评价口蹄疫疫苗质量的关键指标。蔗糖密度梯度离心法（sucrose density gradient centrifugation,SDGC）是公认经典的测定口蹄疫146S抗原含量的方法,但存在检测耗时长、过程复杂、重复性差等缺点,影响了疫苗的质量监测。口蹄疫灭活疫苗146S抗原含量高效液相体积排阻色谱法（size-exclusion high-performance liquid chromatography, SE-HPLC）是一种简便、快速、自动化程度高、高效的测定方法。【目的】 在初步建立的采用SE-HPLC测定口蹄疫灭活疫苗146S抗原含量方法的基础上,针对不同类型、不同浓缩纯化生产工艺制备的口蹄疫灭活疫苗,进一步确认SE-HPLC法在检测过程中的普适性势在必行。【方法】 利用口蹄疫146S抗原标准品建立SE-HPLC法标准曲线,求得回归方程,用于检测样品的146S含量。采用SE-HPLC法和SDGC法分别检测146S抗原标准品1倍、2倍、4倍、8倍、16倍稀释的5个样品和市场上随机选取的22批疫苗, 计算146S抗原含量,对比分析两种方法的相关性。用SE-HPLC法,3次重复检测不同企业生产的134批口蹄疫灭活疫苗146S抗原含量,通过色谱图特异性、检测值相对标准偏差（relative standard deviation ,RSD）分析SE-HPLC法的重复性,评价该方法的适用性,分析市场流通疫苗的总体质量情况。【结果】 SE-HPLC法建立的标准曲线,峰面积与146S抗原含量的线性关系良好（R ²=0.9981,n=8）。口蹄疫146S抗原标准品5个稀释样品和22批口蹄疫疫苗的146S抗原含量检测结果表明,口蹄疫146S抗原含量检测SDGC法和SE-HPLC法高度正相关（R_S ²=0.9994,n_S=5;R_v ²= 0.9602,n_v=22）。134批口蹄疫灭活疫苗中,146S抗原含量相对标准偏差RSD＜5%的疫苗批次分别占疫苗总批次（134批）、单价苗总批次（18批）、双组分及双价苗总批次（76批）、三价苗总批次（40批）的81.34%、72.22%、85.53%、80.00%;146S抗原含量相对标准偏差RSD≤10%的疫苗批次占疫苗总批次（134批）的97.76%。口蹄疫146S抗原含量SE-HPLC法检测重复性良好,其中疫苗146S抗原含量2—4μg·mL ^-1时,检测重复性最好。所有批次疫苗均检测到目的峰,目的峰的平均起峰、最高峰、落峰时间分别为SE-HPLC法进样后的11.58、12.90、14.93min,平均持续3.36min。134批口蹄疫灭活疫苗146S抗原含量为1.11—80.36μg·mL ^-1,其中单价苗、双价苗、双组分苗、三价苗146S抗原含量均值分别为2.07、2.40、2.85、13.14 μg·mL ^-1。【结论】 口蹄疫灭活疫苗146S 抗原含量SE-HPLC法适用性好、重复性高、高效、快速、简便,能够用于检测不同类型的口蹄疫灭活疫苗,从而进行疫苗的质量监测和评估。市场流通的口蹄疫灭活疫苗146S含量较高,疫苗质量较好。

关键词: 高效液相体积排阻色谱法, 口蹄疫灭活疫苗, 146S, 疫苗质量

Abstract:

【Background】 As vaccine manufacturers and regulatory departments, the quality evaluation method of foot-and-mouth disease (FMD) vaccine is particularly important in quality control, where the 146S antigen is the key index. For the quantification of 146S antigen in FMD vaccine, sucrose gradient density centrifugation (SGDC) is recognized as a classical method but the process is complex, time-consuming and poorly repeatable, which affect the quality monitor of vaccines. Size-exclusion high-performance liquid chromatography (SE-HPLC) is previously reported as a simple, rapid, highly automated and efficient technology for analysis of 146S content. 【Objective】 A method of 146S antigen determination in inactivated FMD vaccine by SE-HPLC has been preliminarily established. However, the applicability, repeatability and effectiveness of this method are unknown in the FMD vaccines produced by different manufacturers with different concentration and purification processes. Hence, it is imperative to confirm the universality of the SE-HPLC method.【Method】 Here, the standard curve and regression equation of SE-HPLC method were established with FMD 146S antigen standard, which were used for 146S antigen detection of samples. Moreover, using five samples with 1, 2, 4, 8 and 16 times dilution of 146S antigen standard and 22 batches of vaccine randomly selected in the market, the 146S antigen was detected by SE-HPLC and SDGC respectively, and the correlation between the two methods was analyzed. Further, 134 batches of inactivated FMD vaccines products from different manufacturers in the market were detected three times each for 146S content by SE-HPLC. By analysis of chromatogram specificity of SE-HPLC method and relative standard deviation of 146S content, we confirmed its repeatability and reliability in evaluating all types of vaccine. Finally, 146s content was operated for the quality of FMD vaccines in the market. 【Result】 The standard curve of SE-HPLC method showed good linearity between peak area and concentration of FMD 146S antigen standard (R ²=0.9981, n=8).The detection results of 146S content in 5 diluted 146S antigen standards and 22 batches of FMD vaccine demonstrated that there was a highly positive correlation between SDGC and SE-HPLC methods (R_S ²=0.9994, n_S=5;R_v ²= 0.9602, n_v=22). Among 134 batches of vaccines, the vaccine batches with 146S content relative standard deviation RSD＜5% accounted for 81.34%, 72.22%, 85.53% and 80.00% of the total vaccine batches (134 batches), total monovalent vaccine batches (18 batches), total bicomponent and bivalent vaccine batches (76 batches) and total trivalent vaccine batches (40 batches), respectively. The vaccine batches with 146S content RSD≤10% accounted for 97.76% of the total vaccine batches (134 batches). The SE-HPLC method for detection 146S content in FMD vaccines performed good repeatability, and best repeatability appeared in 2-4μg·mL ^-1146S content of the vaccines. Target peaks could be detected in all batches of vaccine, the average peak starting time, peak and peak falling time of objective chromatographic peaks were 11.58 min, 12.90 min and 14.93 min after HPLC sampling. The average retention of peak was 3.36min. The average 146S content was 1.11-80.36μg·mL ^-1 in all vaccines, 2.07 μg·mL ^-1 in monovalent vaccines, 2.40 μg·mL ^-1 in bivalent vaccines, 2.85 μg·mL ^-1in bicomponent vaccines and 13.14 μg·mL ^-1 in trivalent vaccines respectively.【Conclusion】 These results provided proof that SE-HPLC assay was universal, highly repeatable, reliable, rapid and simple for detection 146S content in various types of current inactivated FMD vaccines in the market, which could be used to monitor and evaluate the quality of the vaccine. FMD vaccines in the market had high 146S content and good vaccine quality.

Key words: size-exclusion high-performance liquid chromatography, inactivated foot-and-mouth disease vaccine, 146S, quality of the vaccine

朱元源,徐嫄,邹兴启,杨延丽,刘丽丽,万建青,李翠,徐璐,张乾义,夏应菊,王兆,郎洪武,王琴,张松平,赵启祖. 高效液相体积排阻色谱法测定口蹄疫灭活疫苗146S 抗原含量及疫苗质量评估[J]. 中国农业科学, 2019, 52(20): 3695-3704.

YuanYuan ZHU,Yuan XU,XingQi ZOU,YanLi YANG,LiLi LIU,JianQing WAN,Cui LI,Lu XU,QianYi ZHANG,YingJu XIA,Zhao WANG,HongWu LANG,Qin WANG,SongPing ZHANG,QiZu ZHAO. Determination of 146S Antigen in Inactivated Foot-and-Mouth Disease Vaccine by Size-Exclusion High-performance Liquid Chromatography and Quality Evaluation of Vaccine[J]. Scientia Agricultura Sinica, 2019, 52(20): 3695-3704.

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参考文献 33

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方法Method	146S抗原含量 Content of 146S（μg·mL^-1）
SE-HPLC	59.50	39.67	19.83	9.91	3.97
SDGC	38.26	26.12	13.64	6.67	2.69

批次Batch	SE-HPLC (μg·mL^-1)	SDGC (μg·mL^-1)	批次Batch	SE-HPLC (μg·mL^-1)	SDGC (μg·mL^-1)
1	10.30+0.01	10.10+0.22	12	85.61+0.05	58.43+3.01
2	10.82+0.06	10.66+1.38	13	2.88+0.00	1.96+0.22
3	7.29+0.02	5.59+0.05	14	1.36+0.02	1.29+0.15
4	8.89+0.01	5.55+0.06	15	2.77+0.00	1.45+0.13
5	6.70+0.03	4.88+0.19	16	2.49+0.03	1.55+0.18
6	7.20+0.01	5.52+0.21	17	2.18+0.01	1.76+0.17
7	15.83+0.02	11.12+1.38	18	2.03+0.01	1.21+0.16
8	59.17+0.02	30.07+2.54	19	2.60+0.02	1.46+0.09
9	44.92+0.02	21.35+1.96	20	2.76+0.00	1.62+0.11
10	8.66+0.07	5.67+0.91	21	2.75+0.14	1.63+0.13
11	24.58+0.02	14.90+1.85	22	10.3+0.01	10.10+1.96

类型 Type	RSD（X） Relative standard deviation	疫苗批次 Batches of vaccine	所占比例 Percentage (%)
疫苗（总） Total batches of vaccines 134批	X＞10%	3	2.24
	5%≤X≤10%	22	16.42
	X＜5%	109	81.34
单价苗 Monovalent vaccine 18批	X＞10%	0.0	0
	5%≤X≤10%	5	27.78
	X＜5%	13	72.22
双组分苗及双价苗 Bivalent and bicomponent vaccines 76批	X＞10%	3	3.95
	5%≤X≤10%	8	10.52
	X＜5%	65	85.53
三价苗 Trivalent vaccines 40批	X＞10%	0	0
	5%≤X≤10%	8	20.00
	X＜5%	32	80.00

疫苗类型及批次 Types and batches of vaccines	146S平均含量 Average content of 146S	146S含量Y Content of 146S (μg·mL^-1)	所占比例(%) Percentage
疫苗类型及批次 Types and batches of vaccines	146S平均含量 Average content of 146S	146S含量Y Content of 146S (μg·mL^-1)	占疫苗批次比例 Percentage of total batches of vaccines	RSD≥5%	RSD＜5%
疫苗总批次 Total batches of vaccines 134批	5.70	Y＞6	22.39	20.00	80.00
		4＜Y≤6	11.19	20.00	80.00
		2≤Y≤4	40.30	11.11	88.89
		Y＜2	26.12	28.57	71.43
单价苗Monovalent vaccines 18批	2.07	2≤Y≤4	61.11	9.09	90.91
单价苗Monovalent vaccines 18批	2.07	＜2	38.89	57.14	42.86
双价苗Bivalent vaccines 37批	2.40	2≤Y≤4	78.38	13.79	86.21
双价苗Bivalent vaccines 37批	2.40	Y＜2	21.62	37.50	62.50
双组分苗 Bicomponent vaccines 39批	2.85	Y＞6	15.38	0.00	0.00
		4＜Y ≤6	7.69	33.33	66.67
		2≤Y≤4	35.90	7.14	92.86
		Y＜2	41.03	18.75	81.25
三价苗 Trivalent vaccine 40批	13.14	Y＞6	60.000	25.000	75.00
		4＜Y≤6	25.000	20.000	80.00
		2≤Y≤4	5.000	0.000	100.00
		Y＜2	10.000	0.000	100.00

高效液相体积排阻色谱法测定口蹄疫灭活疫苗146S 抗原含量及疫苗质量评估

Determination of 146S Antigen in Inactivated Foot-and-Mouth Disease Vaccine by Size-Exclusion High-performance Liquid Chromatography and Quality Evaluation of Vaccine

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