国产重组人粒细胞-巨噬细胞集落刺激因子原液中制品相关蛋白的定性定量分析  

Identification and quantification of product-related proteins in the bulk of recombinant granulocyte-macrophage colony stimulating factor produced in China

作　　者：陶磊[1] 韩春梅[1] 刘兰[1] 史新昌[1] 饶春明[1] TAO Lei;HAN Chun-mei;LIU Lan;SHI Xin-chang;RAO Chun-ming(National Institutes for Food and Drug Control,Key Laboratory of Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京100050

出　　处：《药物分析杂志》2018年第11期1875-1880,共6页Chinese Journal of Pharmaceutical Analysis

基　　金：药品医疗器械审评审批制度改革专项课题(ZG2017-3-02);十三五科技重大专项课题"生物类似药质量相似性评价体系建设研究"(2015ZX09501008)

摘　　要：目的:分析鉴定国产重组人粒细胞-巨噬细胞集落刺激因子原液中的制品相关蛋白,为《中华人民共和国药典》(以下简称《中国药典》)标准提高提供依据。方法:采用欧洲药典方法测定所征集的样品中制品相关蛋白的含量;采用液相质谱联用法对制品相关蛋白进行鉴定,色谱柱为Waters BEH300 C4(2.1 mm×150 mm,1.7μm),以0.1%的三氟乙酸水溶液(A)-0.1%的三氟乙酸90%乙腈溶液(B)为流动相,梯度洗脱,流速0.2 mL·min^(-1)。质谱仪采用Waters公司Xevo G2-S质谱仪,正离子电喷雾离子源,毛细管电压3.0 kV,Cone电压40 V,去溶剂气体温度350℃,去溶剂气体流速800 L·h^(-1),扫描范围(m/z):500～3 000。结果:对4家生产企业的7批原液进行了检测,其中3家企业的5批原液符合欧洲药典标准,2家企业的2批原液不符合欧洲药典标准;相对分子质量测定结果显示,样品中普遍存在的制品相关蛋白为氧化形式及乙酰化形式,个别批次中存在相对分子质量不一的肽段。结论:本次征集的样品有71%符合欧洲药典标准,可以考虑将制品相关蛋白检测纳入新版《中国药典》,以提高国产重组人粒细胞-巨噬细胞集落刺激因子的产品质量。Objective：To quantify and identify the product-related proteins in the bulk of recombinant human granulocyte-macrophage colony stimulating factor（rhGM-CSF） produced in China,and to provide the basis for improving the standard of Chinese Pharmacopoeia. Methods：The European Pharmacopoeia（EP） method was applied to determine the content of product-related proteins in the collected samples. Mass spectrometry combined with ultra performance liquid chromatography was used for the identification. The chromatographic separation was carried out on a Waters BEH300 C4 column（2.1 mm×150 mm,1.7 μm） with the mobile phase consisting of water containing 0.1% trifluoroacetic acid（A）-water containing 0.1% trifluoroacetic acid and 90% acetonitrile（B） in a gradient mode at the flow rate of 0.2 mL·min^（-1）. The mass spectra were obtained by Waters Xevo G2-S mass spectrometer with electrospray ionization source in positive mode. The capillary voltage was 3 kV;Cone voltage was 40 V;the gas temperature was 350 ℃;the gas flow was 800 L·h^（-1） and mass scan range（m/z） was 500~3 000. Results：7 lots of bulk produced by 4 manufacturers were analyzed. 5 lots produced by 3 manufacturers met the EP standard,and the other 2 lots produced by 2 manufacturers were substandard. Results of relative molecular weights showed that the most common product-related proteins were formed by oxidation and acetylation,and peptides with different relative molecular weights were only found in 2 batches. Conclusion：As shown in this study,71% of the collected samples met EP standard. It is reasonable to incorporate the EP method into the new version of Chinese Pharmacopoeia in order to improve the quality of domestic rhGM-CSF.

关 键 词：粒细胞-巨噬细胞集落刺激因子 制品相关蛋白 原液 欧洲药典 质量标准 

分 类 号：R917[医药卫生—药物分析学]