2D-CEX–FcRn–MS to Study Structure/Function Relation of mAb Charge Variants

IF 6.7 1区 化学 Q1 CHEMISTRY, ANALYTICAL
Liesa Verscheure, Isabel Vandenheede, Eline De Rore, Mabelle Meersseman, Valerie Hanssens, Kris Meerschaert, Hilde Stals, Pat Sandra, Frederic Lynen, Filip Borgions and Koen Sandra*, 
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引用次数: 0

Abstract

The automated elucidation of the interplay between monoclonal antibody (mAb) structure and function using two-dimensional liquid chromatography–mass spectrometry (2D-LC–MS) is reported. Charge variants, induced through forced degradation, are resolved by first-dimension (1D) cation-exchange chromatography (CEX) and subsequently collected in loops installed on a multiple heart-cutting valve prior to transfer to second-dimension (2D) neonatal crystallizable fragment receptor (FcRn) affinity chromatography coupled with MS. As such, binding affinity of the latter mAb variants can elegantly be assessed and a first glimpse of identity provided. To maximize MS sensitivity, charge variants are unfolded upon eluting from the 2D affinity column by postcolumn addition of a denaturing solution. Further structural details, i.e., modification sites and chain distribution, are unraveled by a multidimensional LC–MS (mD-LC–MS) setup incorporating 1D CEX and parallel online middle-up and bottom-up LC–MS analysis in the subsequent dimensions. Identified charge variants could be ranked according to their affinity for FcRn. Binding is predominantly impacted by heavy chain (HC) M253 oxidation and to a lesser extend, M429 oxidation. Oxidation of both HCs more drastically affects FcRn interaction compared to single-chain oxidation, and the more oxidation, the less binding. Other modifications, such as HC glycosylation, HC N385/390, and N326 deamidation or HC C-terminal processing, are not shown to affect binding. The streamlined platform is challenged against the established workflow involving offline collection of charge variants and structural and functional assessment by, respectively, LC–MS and enzyme-linked immunosorbent assay (ELISA). A decent correlation is demonstrated between the binding affinity measured with ELISA and 2D FcRn affinity chromatography. In addition, throughput is improved (7-fold), material requirements are substantially reduced (2 orders of magnitude), and sample preparation artifacts and loss are minimized. With the simultaneous determination of mAb structure and function, the current study takes the concept of multiattribute analysis to the next level, thereby contributing to the future development of safer and more effective antibody therapeutics.

Abstract Image

用二维-CEX-FcRn-MS 研究 mAb 电荷变体的结构/功能关系
本研究利用二维液相色谱-质谱联用技术(2D-LC-MS)自动阐明了单克隆抗体(mAb)结构与功能之间的相互作用。通过强制降解诱导的电荷变体由一维(1D)阳离子交换色谱法(CEX)解析,随后收集到安装在多重心脏切割阀上的环路中,再转移到二维(2D)新生儿可结晶片段受体(FcRn)亲和色谱法与质谱联用。因此,可以优雅地评估后一种 mAb 变体的结合亲和力,并初步了解其特性。为了最大限度地提高质谱灵敏度,电荷变体在从二维亲和层析柱洗脱时,会在柱后加入变性溶液使其展开。进一步的结构细节,即修饰位点和链分布,将通过结合一维 CEX 和后续维度的平行在线自上而下和自下而上 LC-MS 分析的多维 LC-MS (mD-LC-MS)装置来揭示。已识别的电荷变体可根据其对 FcRn 的亲和力进行排序。结合主要受重链(HC)M253 氧化的影响,其次是 M429 氧化。与单链氧化相比,这两种 HC 的氧化对 FcRn 的相互作用影响更大,氧化程度越高,结合力越弱。其他修饰,如 HC 糖基化、HC N385/390 和 N326 脱氨基或 HC C 端加工,均未显示会影响结合。简化后的平台面临着既定工作流程的挑战,包括离线收集电荷变体,以及分别通过 LC-MS 和酶联免疫吸附试验(ELISA)进行结构和功能评估。ELISA 和二维 FcRn 亲和层析法测得的结合亲和力之间有很好的相关性。此外,通量提高(7 倍),材料需求大幅减少(2 个数量级),样品制备的假象和损失降至最低。通过同时测定 mAb 的结构和功能,目前的研究将多属性分析的概念提升到了一个新的水平,从而有助于未来开发更安全、更有效的抗体疗法。
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来源期刊
Analytical Chemistry
Analytical Chemistry 化学-分析化学
CiteScore
12.10
自引率
12.20%
发文量
1949
审稿时长
1.4 months
期刊介绍: Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.
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