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.
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.