冻干人用狂犬病疫苗原液纯度体积分子排阻高效液相色谱检测方法的建立及验证

Development and validation of size exclusion column-high performance liquid chromatography for purity determination of bulk material of freeze-dried rabies vaccine for human use

目的 建立冻干人用狂犬病疫苗原液纯度的体积分子排阻高效液相色谱(size exclusion column-high performance liquid chromatography,SEC-HPLC)检测方法,并进行验证。方法 采用TSK-gel G6000PWXL色谱柱(7.8 mm×30 cm,13μm)进行测定,流动相为:0.1 mol/L PB缓冲液(pH 7.8);流速为:0.5 mL/min;检测波长为:280 nm;进样量为:20μL;柱温为:30℃。验证方法的系统适用性、专属性、精密性、耐用性,并确定检测限和定量限。采用建立的方法检测3批冻干人用狂犬病疫苗(Vero细胞)原液及1批冻干人用狂犬病疫苗(人二倍体细胞)原液的纯度。结果 参比品与两种基质制备的冻干人用狂犬病疫苗原液目的蛋白色谱峰的分离度均> 1.5,峰拖尾因子均<1.5;空白溶剂在目的蛋白出峰位置无吸收峰,不干扰测定;精密性验证中的保留时间和峰面积RSD均<2.0%;定量限为10μg/mL,检测限为4μg/mL;参比品在278、280、282 nm 3种不同检测波长下连续进样3次,保留时间和峰面积RSD均<2.0%。4批冻干人用狂犬病疫苗原液的纯度均> 97%。结论 建立的冻干人用狂犬病疫苗原液纯度SECHPLC检测方法具有良好的专属性、精密性及耐用性,为人用狂犬病疫苗的质量控制提供了可靠方法。

Objective To develop and validate size exclusion column-high performance liquid chromatography(SEC-HPLC)for determination of the purity of bulk material of freeze-dried rabies vaccine for human use.Methods Chromatography column TSK-gel G6000PW_(XL)(7.8 mm × 30 cm,13 μm)was used for the determination(column temperature 30 ℃)with mobile phase of 0.1 mol/L PB buffer(pH 7.8)at a flow rate of 0.5 mL/min.The detection wavelength was 280 nm and injection volume was 20 μL.The method was validated for system suitability,specificity,precision and durability and determined for detection limit and quantitation limit,which was applied to analyze bulk material purity of freeze-dried rabies vaccine for human use of 3 batches of Vero cells and 1 batch of human diploid cells.Results The resolution of target protein spectrum peak of bulk material of reference sample and freeze-dried rabies vaccine for human use prepared with two substrates was more than 1.5 with a tailing factor less than 1.5;The blank solvent showed no absorption peak at the position of target protein peak with no interference in the determination;The RSDs of retention time and peak area in precision verification were both less than 2.0%;The quantitative limit was 10 μg/mL,and the detection limit was 4 μg/mL;The reference sample was injected three times continuously at three different detection wavelengths of 278,280 and 282 nm,and the RSDs of retention time and peak area were also less than 2.0%.The purity of 4 batches of freeze-dried rabies vaccine bulk material for human use was all more than 97%.Conclusion The developed SEC-HPLC for determination of the purity of freeze-dried rabies vaccine bulk material for human use showed good specificity,precision and durability,which provided a reliable method for the quality control of human rabies vaccine.