Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan 48109, United States.
Anal Chem. 2021 Dec 7;93(48):16166-16174. doi: 10.1021/acs.analchem.1c03946. Epub 2021 Nov 22.
Ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) assays of monoclonal antibody (mAb)-based biotherapeutics have proven sensitive to disulfide bridge structures, glycosylation patterns, and small molecule conjugation levels. Despite promising prior reports detailing the capabilities of IM-MS and CIU to differentiate biosimilars, generic mAb therapeutics, there remain questions surrounding the sensitivity of CIU to mAb structure changes that occur upon stress, the reproducibility of such measurements across IM-MS platforms, and the correlation between CIU and differential scanning calorimetry (DSC) datasets. In this report, we describe a comprehensive IM-MS and CIU dataset acquired for three Infliximabs: Remicade, Inflectra, and Renflexis. We subject each infliximab sample to forced degradation through heat stress and observe broadly similar yet subtly different stability patterns for these three biotherapeutics. We find that CIU is capable of tracking differences in mAb higher-order structure (HOS) imparted during forced heat stress degradation and that DSC is less sensitive to these alterations in comparison. Furthermore, we collected our comprehensive IM-MS and CIU data across two instrument platforms (Waters G2 and Agilent 6560), with both producing similar abilities to differentiate mAbs while also revealing minor differences between the results obtained on the two instruments. Finally, we demonstrate that CIU-based heatmaps and classification allow for rapid assessment of the most differentiating charge states for the analysis of infliximab, and using multiplexed classification, we conservatively estimate a 30-fold improvement in the time required to perform mAb stability and HOS measurements over standard DSC tools.
离子淌度-质谱（IM-MS）和碰撞诱导 unfolding（CIU）分析已被证明对单克隆抗体（mAb）生物治疗药物的二硫键结构、糖基化模式和小分子缀合水平敏感。尽管有先前的报告详细描述了 IM-MS 和 CIU 区分生物类似物、通用 mAb 治疗药物的能力，但仍存在一些问题，例如 CIU 对 mAb 结构在应激下发生变化的敏感性、在 IM-MS 平台之间进行此类测量的重现性以及 CIU 与差示扫描量热法（DSC）数据集之间的相关性。在本报告中，我们描述了针对三种英夫利昔单抗（Remicade、Inflectra 和 Renflexis）获得的全面 IM-MS 和 CIU 数据集。我们通过热应激使每个英夫利昔单抗样品强制降解，并观察到这三种生物治疗药物具有广泛相似但微妙不同的稳定性模式。我们发现，CIU 能够跟踪在强制热应激降解过程中赋予 mAb 的高阶结构（HOS）的差异，而 DSC 对这些变化的敏感度较低。此外，我们在两个仪器平台（沃特世 G2 和安捷伦 6560）上收集了全面的 IM-MS 和 CIU 数据，这两个平台都具有区分 mAb 的相似能力，同时还揭示了两个仪器之间结果的细微差异。最后，我们证明了基于 CIU 的热图和分类允许快速评估分析英夫利昔单抗最具区分能力的电荷状态，并且使用多路分类，我们保守估计与标准 DSC 工具相比，进行 mAb 稳定性和 HOS 测量所需的时间可提高 30 倍。
