Absolute Quantitation of Coeluting Impurities in Peptide Drugs Using High Resolution Mass Spectrometry: Glucagon a Case Study in Pharmaceutical Development

IF 2.7 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Anusha Kodidasu, , , Koushik Acharyya*, , and , Vasanthakumar Ganga Ramu*, 
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Abstract

Synthetic peptide-based drugs provide customized therapeutic solutions, but developing a peptide medicine presents various challenges, especially in terms of impurity management. This holds true when traditional techniques like RP-HPLC fail to separate low-abundance coeluting impurities. In this regard, liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) has proven to be effective for identifying and characterizing peptide impurities, although its application for accurate quantitation is still limited. This study developed and validated two quantitation strategies using UPLC-HRMS, employing full scan and pseudo-MRM (p-MRM) modes, to identify and accurately quantify two structurally similar coeluting peptide impurities (des-Gly4 glucagon and des-Thr5 glucagon) observed in synthetic glucagon. The methods exhibited high specificity and demonstrated good linearity (R2 > 0.99) across a concentration range of 0.25–25 μg/mL. The limits of detection (LODs) for des-Gly4-glucagon and des-Thr5-glucagon were determined to be as low as 0.01% and 0.02%, with limits of quantitation (LOQs) at 0.02% and 0.04%, respectively. Precision (RSD%) was recorded at less than 10%, and recovery ranged from 100% to 120%. A comparative analysis of the results indicated that both quantifitation methods performed similarly in terms of accuracy, precision, and recovery, producing comparable impurity estimates in three glucagon API samples. However, p-MRM showed slightly better linearity and sensitivity compared to the full scan EIC-based quantitation method.

Abstract Image

利用高分辨率质谱法对多肽药物中溶出杂质的绝对定量:胰高血糖素——药物开发中的一个案例研究。
合成多肽药物提供了定制的治疗方案,但开发多肽药物面临各种挑战,特别是在杂质管理方面。当RP-HPLC等传统技术无法分离低丰度的溶出杂质时,这一点是正确的。在这方面,液相色谱结合高分辨率质谱(LC-HRMS)已被证明是鉴定和表征肽杂质的有效方法,尽管其在精确定量方面的应用仍然有限。本研究开发并验证了UPLC-HRMS的两种定量策略,采用全扫描和伪mrm (p-MRM)模式,以鉴定和准确定量合成胰高血糖素中观察到的两种结构相似的糖化肽杂质(des-Gly4胰高血糖素和des-Thr5胰高血糖素)。该方法在0.25 ~ 25 μg/mL范围内具有较高的特异性和良好的线性(R2 > 0.99)。des- gly4 -胰高血糖素和des- thr5 -胰高血糖素的检测限(lod)分别为0.01%和0.02%,定量限(loq)分别为0.02%和0.04%。精密度(RSD%)小于10%,回收率为100% ~ 120%。结果的比较分析表明,两种定量方法在准确度、精密度和回收率方面表现相似,在三种胰高血糖素原料药样品中产生可比较的杂质估计。然而,与基于全扫描eic的定量方法相比,p-MRM显示出略好的线性和灵敏度。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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