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

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，1.7 μm），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.