Jing Xu, John E Coughlin, Malgorzata Szyjka, Serene Jabary, Sonal Saluja, Zoran Sosic, Yunqiu Chen, Chong-Feng Xu
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引用次数: 0
摘要
聚集物被认为是单克隆抗体(mAb)治疗药物中最关键的产品相关杂质之一,因为它们会对药物的稳定性和安全性产生负面影响。迄今为止,研究工作主要集中于了解 mAb 自身聚集的原因和影响,包括内部和外部因素。在本研究中,我们重点了解通过铰链裂解和损失一个 Fab 单元(简称为 "Fab/c")形成的单价片段在 mAb 存在的情况下的稳定性。Fab/c 片段是通过有限的 IgdE 消化产生的,这种消化能特异性地裂解 IgG1 mAb 铰链区上方,然后进行疏水相互作用色谱 (HIC) 富集。在热加速条件下孵育两种含有不同程度 Fab/c 片段的 IgG1 mAb。开发并使用了一种基于尺寸排阻色谱结合原位质谱(SEC-UV-native MS)的方法来表征稳定性样品,并确定了异质二聚体的形成,包括完整二聚体、mAb-Fab/c 二聚体、Fab/c-Fab/c 二聚体和 mAb-Fab 二聚体。对聚集动力学的定量分析表明,糖基化 mAb(mAb1)和非糖基化 mAb(mAb2)之间,Fab/c 片段对单个二聚体聚集率的影响是不同的。在 25°C 加速稳定条件下对脱糖 mAb1 进行的另一项研究表明,N-聚糖对 mAb1 的总聚集率没有显著影响。这项研究还凸显了 SEC-UV-native MS 方法在表征 mAb 样品中分离、鉴定和量化 mAb 聚集体和片段方面的能力。
Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry.
Aggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as "Fab/c"), a commonly observed impurity during manufacturing and stability. The Fab/c fragments were generated using a limited IgdE digestion that specifically cleaves above the IgG1 mAb hinge region, followed by hydrophobic interaction chromatographic (HIC) enrichment. Two IgG1 mAbs containing different levels of Fab/c fragments were incubated under thermally accelerated conditions. A method based on size exclusion chromatography coupled with native mass spectrometry (SEC-UV-native MS) was developed and used to characterize the stability samples and identified the formation of heterogeneous dimers, including intact dimer, mAb-Fab/c dimer, Fab/c-Fab/c dimer, and mAb-Fab dimer. Quantitative analyses on the aggregation kinetics suggested that the impact of Fab/c fragment on the aggregation rate of individual dimer differs between a glycosylated mAb (mAb1) and a non-glycosylated mAb (mAb2). An additional study of deglycosylated mAb1 under 25°C accelerated stability conditions suggests no significant impact of the N-glycan on mAb1 total aggregation rate. This study also highlighted the power of SEC-UV-native MS method in the characterization of mAb samples with regard to separating, identifying, and quantifying mAb aggregates and fragments.
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.