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期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237
 

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重组人粒细胞刺激因子的N端氨基酸序列异质性分析

Analysis of N-terminal amino acid sequence heterogeneity of recombinant human granulocyte colony-stimulating factor

作者(英文):
分类号:R917
出版年·卷·期(页码):2018,38 (11):1887-1892
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

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。

-----英文摘要:---------------------------------------------------------------------------------------

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.

-----参考文献:---------------------------------------------------------------------------------------

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