Development and verification of a reversed phase high-performance liquid chromatography method for purity determination of recombinant Mycobacterium tuberculosis Ag85b protein stock solution
10.13200/j.cnki.cjb.004138
- VernacularTitle:重组结核分枝杆菌Ag85b蛋白原液纯度反向高效液相色谱检测方法的建立及验证
- Author:
JIN Liting
- Publication Type:Journal Article
- Keywords:
Reversed phase high-performance liquid chromatography;
Mycobacterium tuberculosis(Mtb);
Ag85b protein;
Purity
- From:
Chinese Journal of Biologicals
2024;37(1):86-91
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop and verify a reversed phase high-performance liquid chromatography method for the determination of the purity of recombinant Mycobacterium tuberculosis(Mtb)Ag85b protein stock solution.Methods Fourfactor,three-level orthogonal test was designed,with the area,trailing factor,peak area and peak area RSD as the evaluation indexes to explore the optimal detection conditions. The methodology verification of specificity,linear range,precision and durability was conducted in accordance with the general principles of Chinese Pharmacopoeia(Volume Ⅳ,2020 edition)9101.Results The results of all the evaluation indexes were good when the elution ratio of organic phase was30% ～ 95%,the detection temperature was 35 ℃,the sample volume was 3 μg,and the elution time of 95% organic phase was 15 min. The method had the linear correlation coefficient(R2)of 0. 998 5,the linear range of 1. 8 ～ 4. 2 μg,the reproducibility RSD of 0. 01%,and the intermediate precision RSD of 0. 16%,with good durability under slight changes of column temperature and flow rate.Conclusion The reversed phase high-performance liquid chromatography method for the purity determination of recombinant Mtb Ag85b protein stock solution was developed,which has good specificity,precision and durability,and can be used for the quality control of recombinant Mtb Ag85b protein stock solution.
