目的 解析合成多肽AAT002原料药中2个双环未知降解杂质及4个同位点未知水解杂质结构。方法 分析多肽侧链基团亲核进攻能力,根据杂化轨道理论,提出碳负离子消旋机制和亚稳态分子内重排机制,利用高分辨质谱二级碎片和Proteomics toolkit预测模型,匹配理论和实测B+、Y+碎片,验证6个杂质的结构。结果 匹配结果验证了碳负离子消旋机制和亚稳态分子内重排机制,建立了三级杂质降解关联网络,分析了减少杂质生成的条件。结论 碳负离子的形成是杂质降解网络的关键点,在纯化和贮存中减少促进碳负离子形成的弱碱性条件,更加有利于合成多肽冻干、储存。
Abstract
OBJECTIVE To analyze the structures of two unknown bicyclic degradation impurities and four unknown iso-site hydrolytic degradation impurities in the synthetic peptide AAT002 API. METHODS The nucleophilicity of side chain groups of polypeptides was analyzed. Based on the hybrid orbital theory,the carbon anion racemization mechanism and the metastable intramolecular rearrangement mechanism were proposed. Using MS/MS fragments of high resolution mass spectrometry and proteomics toolkit fragment prediction model,the theoretical fragments and measured fragments were matched. And the structures of six impurities were verified. RESULTS The matching results verified the proposed carbon anion racemization mechanism and metastable intramolecular rearrangement mechanism. A three-level correlation network for impurity degradation was established. The conditions for reducing the formation of impurities were analyzed. CONCLUSION The formation of carbon anions is the key point of impurity degradation network. The weak alkaline conditions that promote the formation of carbon anions should be reduced during purification and storage. More attention should be paid to the changes in freeze-drying and storage conditions.
关键词
亚稳态分子内重排 /
降解杂质 /
关联网络 /
多肽
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Key words
metastable intramolecular rearrangement /
degradation impurity /
correlation network /
peptide
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中图分类号:
R917
Q591
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脚注
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