HPLC在注射用重组人粒细胞巨噬细胞刺激因子冻干粉针剂主药含量测定中应用.docVIP

HPLC在注射用重组人粒细胞巨噬细胞刺激因子冻干粉针剂主药含量测定中应用 　　[摘要]目的 建立注射用重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF）冻干粉针剂中主要活性成分的HPLC的测定方法。方法 HPLC色谱条件中，色谱柱为TSK gel G3000SW（300.0 mm×7.5 mm，5 μm），流动相为0.1 mol/L磷酸盐缓冲液，流速为1 ml/min，自动进样20～40 μl，检测波长为280 nm。研究该色谱条件是否能分析rhGM-CSF冻干粉针剂中的各组分、实验重现性以及不同进样体积与各组分峰面积的线性关系。结果 检测出rhGM-CSF冻干粉针剂中的主成分为rhGM-CSF蛋白和人血白蛋白，rhGM-CSF蛋白进样量与峰面积呈良好线性关系，R20.99，百分含量的RSD值均3%。结论 该法可用于检测rhGM-CSF冻干粉针剂蛋白量在10～20 μg范围内的的活性成分含量。 　　[关键词]重组人粒细胞巨噬细胞集落刺激因子；冻干粉针剂；高效液相色谱法 　　[中图分类号] R927.2 [文献标识码] A [文章编号] 1674-4721（2017）05（c）-0104-04 　　[Abstract]Objective To establish the method of the main active ingredients in recombinant human granulocyte macrophage colony stimulating factor freeze-dried powder injection （rhGM-CSF） by HPLC.Methods The chromatographic condition of HPLC was chromatographic column （TSK gel G3000SW，300.0 mm×7.5 mm，5 μm），Mobile phase was 0.1 mol/L phosphate buffer，Flow rate was 1 ml / min，automatic sampling was 20-40 μl and detection wavelength was 280 nm.Analyzing the ingredients of rhGM-CSF freeze dried powder injection，reproducibility and linear relationship between different sample volume and peak area of each component under this chromatographic condition were studied in this experiment.Results The main components of rhGM-CSF freeze dried powder injection were rhGM-CSF protein and human albumin.The rhGM-CSF protein sample volume and peak area were good linear （R20.99） and the percentage content of RSD values was less than 3%.Conclusion This method could be used to detect the active component content of the rhGM-CSF lyophilized powder for injection protein with 10-20 μg scope. 　　[Key words]Recombinant human granulocyte macrophage colony stimulating factor；Freeze-dried powder injection；High performance liquid chromatography 　　重?M人粒细胞巨噬细胞刺激因子冻干粉针剂（rhGM-CSF）是使用重组基因工程技术生产的重组蛋白药物[1]343-344，是一种细胞因子，主要用于治疗因放、化疗引起的白细胞减少[2-5]，并且临床上可以治疗溃疡、烫伤、烧伤等创面疾病[6-9]，其活性成分为重组人粒细胞巨噬细胞刺激因子，该品种已被《中国药典》三部收载[1]343-344。《中国药典》未规定其成品的含量测定方法，只要求测定生物学活性（效价），采用的是TF-1依赖细胞株，用MTT染色法进行测定，合格范围是标示活性的80%～150%[10]96