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

Development and verification of a reversed phase high-performance liquid chromatography method for purity determination of recombinant Mycobacterium tuberculosis Ag85b protein stock solution

目的 建立检测重组结核分枝杆菌(Mycobacterium tuberculosis,Mtb)Ag85b蛋白原液纯度的反向高效液相色谱法,并进行验证。方法 设计4因素3水平正交试验,将面积、拖尾因子、峰面积、峰面积RSD作为评价指标进行分析,摸索最适检测条件,并依据《中国药典》四部(2020版)9101药品质量标准分析方法验证指导原则进行专属性、线性范围、精密度、耐用性等验证。结果 当检测条件为有机相洗脱比例30%~95%、检测温度35℃、进样量3μg、95%有机相洗脱时间15 min时,各评价指标结果均较好,在该检测条件下线性相关系数(R2)为0.998 5,线性范围为1.8~4.2μg;重复性RSD为0.01%;中间精密度RSD为0.16%;在柱温与流速细微变动下均具有良好的耐用性。结论 建立了重组MTBAg85b蛋白原液纯度检测的反向高效液相色谱法,该方法专属性强,精密度、耐用性好,可用于重组MTBAg85b蛋白原液质量控制。

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.