Development and verification of a size-exclusion high-performance liquid chromatography method for purity detection of Coxsackievirus A10 vaccine bulk solution
10.13200/j.cnki.cjb.004638
- VernacularTitle:柯萨奇病毒A10疫苗原液纯度尺寸排阻高效液相色谱检测方法的建立及验证
- Author:
Anna YANG
1
Author Information
1. Wuhan Institute of Biological Products Co., Ltd., National Key Laboratory for Novel Vaccines Research and Development of Emerging Infectious Diseases, Hubei Provincial Vaccine Technology Innovation Center, Wuhan 430060, Hubei Province, China
- Publication Type:Journal Article
- Keywords:
Coxsackievirus A10(CVA10) vaccine;
Bulk solution;
Purity;
Size-exclusion high-performance liquid chromatography(SE-HPLC)
- From:
Chinese Journal of Biologicals
2026;39(01):78-84+92
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a size-exclusion high-performance liquid chromatography(SE-HPLC) method for determining the purity of Coxsackievirus A10(CVA10) vaccine bulk solution and to verify and apply the method.Methods The chromatographic conditions were as follows: The mobile phase was 5 mmol/L PBS with 150 mmol/L NaCl(pH 7. 20), and the detection wavelength was 280 nm; the injection volume was 100 μL at the column temperature of 28 ℃. The chromatographic columns[TSKgel G5000 PWXL(7. 8 mm × 300 mm), TSKgel GMPWXL(7. 8 mm × 300 mm), ZenixSEC-300(7. 8 mm ×300 mm), and MonomixMC10-SEC(7. 8 mm × 300 mm)]and flow rates(0. 4, 0. 6 and 0. 8 mL/min) were screened.Subsequently, the specificity, repeatability, intermediate precision, linear range and robustness of the method were verified and the limit of detection(LOD) and limit of quantitation(LOQ) were determined. The optimized method was used to monitor the stability of samples in vaccine bulk solution purification process and CVA10 antigen at 4 ℃.Results The column with the best separation was TSKgel G5000 PWXL(7. 8 mm × 300 mm), and the optimal detection flow rate was 0. 6 mL/min. The method could completely separate the virus antigen peak from other impurity peaks. The relative standard deviations(RSDs)of retention time, peak area and peak height in repeatability verification were 0. 05%, 0. 35% and 0. 13%, respectively, while the RSDs of those in intermediate precision verification were 0. 07%, 0. 81% and 0. 34%, respectively. The peak area showed a good linear relationship with the sample protein concentration, with a regression equation of y = 26. 77 x + 6. 00, R~2= 0. 999.The LOQ was 0. 125 μg/mL and the LOD was 0. 031 μg/mL. This method could tolerate the column temperature ranging from 24 to 32 ℃ and the mobile phase pH values ranging from 7. 0 to 7. 4.Conclusion The established SE-HPLC method has good specificity, repeatability, intermediate precision, linear range and robustness, which is suitable for the detection of virus antigen purity in CVA10 vaccine bulk solution.
