Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (20): 3695-3704.doi: 10.3864/j.issn.0578-1752.2019.20.019

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

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. 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 E-mail:zhaoqizu@163.com

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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 (RS 2=0.9994, nS=5;Rv 2= 0.9602, nv=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

Fig. 1

Calibration curve for qualification of inactivated FMDV 146S antigen"

Fig. 2

Chromatograms of FMDV 146S antigen standard"

Fig. 3

Chromatograms of FMDV 146S antigen standard"

Table 1

Comparison of FMDV 146S antigen standard by SE- HPLC and SDGC"

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

Table 2

Comparison of 146S content of FMDV vaccines by SE-HPLC and SDGC"

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

Table 3

Repeatability analysis of 146S content of inactivated FMDV vaccine by SE-HPLC"

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

Table 4

146S antigen content of inactivated FMDV vaccine and detection analysis"

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
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
＜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
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

Fig. 4

Typical chromatograms of inactivated FMDV vaccine by SE-HPLC Before means before processing by Benzonase;After means after processing by Benzonase"

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