重组甘精胰岛素相关杂质的结构确认及质量分析

Structure Identification and Quality Analysis of Relative Impurities of Recombinant Insulin Glargine

目的结合国际通用标准,通过对甘精胰岛素主要相关杂质的定性研究和来源分析,促进国内同类产品的质量提高。方法首先将提纯冻干的杂质回加到对照品中进行液相色谱定位,继而分析HPLC肽图,结合激光辅助基质解吸附飞行时间质谱测定的分子量及肽质量指纹图谱初步定性,再以小分子电泳、N-端测序及二级质谱进一步验证。结果主要相关杂质均为甘精胰岛素的类似物,是在酶切过程中产生的副产物,即为可预期的工艺相关杂质。结论该方法可对甘精胰岛素的主要相关杂质快速、经济、准确的定性,本实验对预期杂质的来源分析可为同类产品的杂质研究提供新颖的参考思路。

OBJECTIVE To improve the quality of insulin glargine by identifying primary impurities and their source. METHODS Firstly, the purified impurities were spiked in the standard of insulin glargine and were determined by HPLC. Secondly, major impurities were indentified by HPLC peptide map. Molecular weight and peptide mass fingerprinting with obtained by MALDITOF, and validated by tricine SDS-PAGE, N-terminal sequencing and mass/mass spectrometry. RESULTS Major impurities were analogs of insulin glargine and relative to the enzymatic steps in the manufacture process. CONCLUSION This method is rapid, economical and accurate for identifying primary impurities of insulin glargine. The novel idea in analyzing the source of impurities can be a reference for the study of impurities in other insulin analogs.