在单克隆抗体的非还原肽图谱表征中克服人工二硫扰的实用解决方案。

IF 5.6 2区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL
mAbs Pub Date : 2024-01-01 Epub Date: 2024-10-26 DOI:10.1080/19420862.2024.2420805
Andrew Kleinberg, Yuan Mao, Ning Li
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引用次数: 0

摘要

通过分离特定半胱氨酸之间的二硫连接,非还原肽图谱为鉴定治疗性单克隆抗体提供了重要数据。然而,生物制药行业中使用的传统消化策略已被证明会无意中造成二硫连接的重新排列(二硫扰乱),从而生成不能准确代表所分析蛋白质的连接图谱。常见的误解(如避免碱性-pH消化以防止二硫扰断)导致了替代试剂和条件的开发,这些试剂和条件可以缓解这一问题,但却产生了有问题的消化图谱。在此,我们系统而全面地研究了准确的非还原肽图谱绘制的主要考虑因素,并提供了有效而实用的解决方案,以尽量减少不受欢迎的行为,同时仍能获得高质量的消化物。此外,我们还介绍了一种利用有意二硫扰乱作为参考工具的方法,以证明我们提出的策略的稳健性。我们还介绍了作为半胱氨酸烷基化试剂的马来酰亚胺,并展示了它在与 N-乙基马来酰亚胺等业界领先的类似物相兼容方面的优势。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Practical solutions for overcoming artificial disulfide scrambling in the non-reduced peptide mapping characterization of monoclonal antibodies.

Non-reduced peptide mapping provides essential data for characterizing therapeutic monoclonal antibodies by isolating disulfide connections between specific cysteines. However, conventional digestive strategies used throughout the biopharmaceutical industry have been shown to cause unintentional rearrangement of disulfide connections (disulfide scrambling), thus generating connectivity profiles that do not accurately represent the protein being analyzed. Common misconceptions (e.g. avoiding basic-pH digestion to prevent disulfide scrambling) have led to the development of alternative reagents and conditions that can alleviate this issue, but yield problematic digestion profiles. Herein, we systematically and comprehensively examine the primary considerations for accurate non-reduced peptide mapping, and provide effective, practical solutions to minimize undesired behavior while still yielding high-quality digests. Additionally, we present a method that exploits intentional disulfide scrambling as a reference tool to demonstrate the robustness of our proposed strategies. We also introduce maleimide as a cysteine-alkylating reagent and demonstrate its benefits over industry-leading analogs such as N-ethylmaleimide in terms of compatibility with regulatory reports.

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来源期刊
mAbs
mAbs 工程技术-仪器仪表
CiteScore
10.70
自引率
11.30%
发文量
77
审稿时长
6-12 weeks
期刊介绍: mAbs is a multi-disciplinary journal dedicated to the art and science of antibody research and development. The journal has a strong scientific and medical focus, but also strives to serve a broader readership. The articles are thus of interest to scientists, clinical researchers, and physicians, as well as the wider mAb community, including our readers involved in technology transfer, legal issues, investment, strategic planning and the regulation of therapeutics.
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