An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping
An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping
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DOI：
10.1016/j.xphs.2019.07.019
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发表时间：
2019-11-01
影响因子：
3.8
通讯作者：
Wang, Jihong
中科院分区：
文献类型：
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作者：
Cao, Mingyan;Xu, Weichen;Wang, Jihong
mAb经历几种翻译后修饰，包括由天冬酰胺的脱酰胺或天冬氨酸的异构化形成琥珀酰亚胺。由于琥珀酰亚胺形成对mAb的生物活性的潜在影响，该物质的检测和定量是制药行业关注的关键领域。然而，评估琥珀酰亚胺稳定性的研究有限，开发的监测琥珀酰亚胺的方法要么是产品特异性的，要么不耐用。在这里，我们报告了一种平台低pH肽图方法的开发，该方法使用低pH抗性Lys-C和修饰的胰蛋白酶的组合来维持琥珀酰亚胺稳定性，消除脱酰胺测定伪影，并在碱性条件下实现与传统胰蛋白酶肽图方法等效的有效mAb消化。使用该方法，在体外研究中准确评估了琥珀酰亚胺在血清中的稳定性，并确定半衰期为1.5天。由于患者可能暴露于琥珀酰亚胺中间体，因此开发了一种可靠的方法来测量位点特异性脱酰胺和琥珀酰亚胺中间体。再加上一个四极杆质量检测器，我们的方法是自动化的消化数据处理和适用于良好的生产实践环境。对该方法进行了充分确认，以证明准确度、精密度、线性和耐用性。(c)2019年，任作家。爱思唯尔公司出版美国药学协会（American Pharmacopoeia Association）（R）这是一个在CC BY-NC-ND许可证下的开放获取文章（http：//creativecommons.org/licenses/by-nc-nd/4.0/）。
mAbs undergo several post-translational modifications, including the formation of succinimide from the deamidation of asparagine or the isomerization of aspartic acid. Because of the potential impact of succinimide formation on the biological activity of mAbs, detection and quantification of this species is a key area of interest for the pharmaceutical industry. However, studies assessing succinimide stability have been limited, and methods developed to monitor succinimide are either product specific or not robust. Here, we report the development of a platform low-pH peptide-mapping method using a combination of low-pH-resistant Lys-C and modified trypsin to maintain succinimide stability, eliminate deamidation assay artifact, and achieve efficient mAb digestion equivalent to conventional tryptic peptide-mapping method under alkaline condition. Using this method, succinimide stability in serum was accurately assessed in vitro study and the half-life was determined to be 1.5 days. With potential patient exposure to succinimide intermediate, a reliable method was developed to measure site-specific deamidation and succinimide intermediate. Coupled with a single quadrupole mass detector, our method was automated from digestion to data processing and applicable in a good manufacturing practice environment. The method was fully qualified to demonstrate accuracy, precision, linearity, and robustness. (c) 2019 The Authors. Published by Elsevier Inc. on behalf of the American Pharmacists Association (R). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).