药学学报, 2021, 56(9): 2352-2359
引用本文:
黄露, 刘博, 范慧红. 基于质谱分析技术的科博肽中脱酰胺杂质测定方法研究[J]. 药学学报, 2021, 56(9): 2352-2359.
HUANG Lu, LIU Bo, FAN Hui-hong. Determination method of deamidation impurities in cobratide using mass spectrometry technique[J]. Acta Pharmaceutica Sinica, 2021, 56(9): 2352-2359.

基于质谱分析技术的科博肽中脱酰胺杂质测定方法研究
黄露1,2#, 刘博1,2#, 范慧红1,2*
1. 中国食品药品检定研究院, 北京 100050;
2. 国家药品监督管理局化学药品质量研究与评价重点实验室, 北京 102629
摘要:
脱酰胺是蛋白多肽类药物中最为常见的降解杂质来源之一。蛋白序列中谷氨酰胺和天冬酰胺经脱酰胺降解后会导致蛋白化学与生物学性质的改变。质谱分析技术可用于定量蛋白及多肽中的脱酰胺杂质,然而传统的基于胰蛋白酶的bottom-up策略,酶切过程中易人为引入脱酰胺,往往导致脱酰胺杂质的测定结果偏高。本研究通过对Glu-C酶酶解条件的优化,获得了在酸性条件下的最佳酶解条件,显著降低了酶切过程造成的人为引入脱酰胺的现象,鉴定了科博肽样品中的脱酰胺位点(N48)。并进一步通过方法学考察与不同方法的测定结果对比,验证了方法的专属性、重复性与准确性。本研究所建方法为科博肽及其相似蛋白多肽类生化药品中脱酰胺杂质的精准测定奠定了坚实的基础。
关键词:    科博肽      Glu-C酶      脱酰胺      精准测定      生物质谱     
Determination method of deamidation impurities in cobratide using mass spectrometry technique
HUANG Lu1,2#, LIU Bo1,2#, FAN Hui-hong1,2*
1. National Institutes for Food and Drug Control, Beijing 100050, China;
2. NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs, Beijing 102629, China
Abstract:
Deamidation is one of the most common degradation impurities in protein and peptide drugs. The deamidation of glutamine and asparagine in the protein sequence can lead to changes in the chemical and biological properties of the protein. However, the rutine trypsin-based pretreatment process can significantly increase the artificial deamidation impurities during the digestion process, resulting in high determination level. In this study, after optimizing the conditions of Glu-C enzymatic hydrolysis, we obtained the best enzymatic conditions under acidic condition and the artificial deamidation impurities significantly reduced in digestion process, identified the deamidation site (N48). Through the methodological investigation and comparison of the measurement results of different methods, the specificity, reproducibility and accuracy of the method are verified. The method established in this research has laid a solid foundation for the accurate determination of deamidation impurities in cobratide and its similar protein peptide biochemical drugs.
Key words:    cobratide    Glu-C enzyme    deamidation    accurate determination    biological mass spectrometry   
收稿日期: 2021-04-25
DOI: 10.16438/j.0513-4870.2021-0619
基金项目: 北京市自然科学基金资助项目(7194337);国家药品监督管理局化学药品质量研究与评价重点实验室课题-蛋白及多肽类生化药物质量评价关键技术研究(1030050090132).
通讯作者: 范慧红,Tel:86-10-53851585,E-mail:shenghuayaoshi@126.com
Email: shenghuayaoshi@126.com
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