重组人粒细胞刺激因子的N端氨基酸序列异质性分析

目的：分析重组人粒细胞刺激因子（rhG-CSF）的N端异质性。方法：采用反相超高效液相色谱法分离rhG-CSF中N端不一致的各组分；色谱条件：采用Waters BEH 300 C₄（2.1 mm×100 mm，1.7 μm）色谱柱，以0.1%三氟乙酸水溶液（A）-0.1%三氟乙酸乙腈溶液（B）为流动相，梯度洗脱，流速0.2 mL·min^-1。分别收集各组分并冷冻干燥，采用N端测序仪测定各组分的N端氨基酸序列；液质联用测定rhG-CSF的相对分子质量，对N端测序结果进行验证质谱仪采用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，锥孔电压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：色谱分离得到3个组分；N端氨基酸序列测定结果显示，3个组分分别为rhG-CSF主成分及2个N端异质性相关蛋白；质谱相对分子质量测定结果与N端测序结果一致。结论：应用色谱法及质谱法可对rhG-CSF中的N端异质性相关蛋白进行分析检测，检测结果可为国产rhG-CSF制品的质量控制和标准提升提供参考和数据支持。

Objective: To analyze the N-terminal heterogeneity of recombinant human granulocyte colony-stimulating factor (rhG-CSF). Methods: The N-terminal inconsistent components in rhG-CSF were separated by reversed phase ultra high performance liquid chromatography. The chromatographic separation was carried out on a Waters BEH 300 C₄ column(2.1 mm×100 mm,1.7 μm) with the mobile phase consisting of 0.1% trifluoroacetic acid aqueous solution (A)-0.1% trifluoroacetic acid acetonitrile solution (B) in a gradient mode at the flow rate of 0.2 mL·min^-1. Each component was collected and freeze-dried,and the N-terminal amino acid sequences of them were determined by N-terminal sequencer. The relative molecular mass of rhG-CSF was measured by liquid chromatography mass spectrometry so as to verify the results of N-terminal sequencing. Waters Xevo G2-S mass spectrometer with electrospray ionization source in positive mode was used,the capillary voltage was 3 kV,Cone voltage was 40 V,gas temperature was 350,gas flow was 800 L·h^-1,mass scan range was m/z 500-3 000. Results: Three components were separated by chromatography,and the results of N-terminal amino acid sequence analysis showed that the three components were rhG-CSF principal components and two N-terminal heterogeneity related proteins,respectively. The results of relative molecular weight determination were consistent with N-terminal sequencing. Conclusion: N terminal heterogeneity of rhG-CSF can be analyzed and detected by chromatography and mass spectrometry,which could provide reference and data support for quality control and standard improvement of domestic rhG-CSF products.