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Analytica Chimica Acta

Analytica Chimica Acta

IF: 5.7
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Sequencing the monoclonal antibody variable regions using multiple charge integration middle-down strategy and ultraviolet photodissociation

Published:19 November 2024 DOI: 10.1016/j.aca.2024.343450
Jialiang Liu , Zheyi Liu , Heng Zhao , Chunlei Xiao , Xueming Yang , Fangjun Wang

Abstract

Background

Therapeutic monoclonal antibodies (mAbs) have become essential biopharmaceuticals for clinical targeted therapies due to their high specificity, affinity and low side effects. The specificity and affinity of mAb to targeting antigen are mainly dependent on the three complementarity determining regions (CDRs) with high variations in amino acid sequences. Therefore, mAb CDR sequencing is crucial for the characterization of therapeutic mAbs. Here, we developed a 193-nm ultraviolet photodissociation (UVPD) based multiple charge integration middle-down mass spectrometry (MCI-MDMS) strategy for mAb sequencing.

Results

We demonstrate that the UVPD spectra of mAb subunit ions with different charge states exhibit high complementarity, and integration can result in higher sequence coverage compared to single charge states. Finally, over 95?% sequence coverage of two different mAbs has been achieved with full sequence coverage of CDRs, underscoring the great potential of this strategy in accurate sequencing of mAb variable regions. Compared with the conventional higher energy collisional dissociation (HCD) strategy of mAb subunit sequencing, the sequence coverage of CDRs at single UVPD subunit charge state has increased by an average of 30?%. In addition, almost complete sequence coverage of mAb ensures the accurate localization of mAb post-translational modifications (PTMs), including glycosylation of two different sites, C-terminal lysine truncation, and N-terminal cyclization of glutamine.

Significance

The integration of MCI-MDMS and UVPD realizes high sequence coverage and reliable PTM determination of mAbs. This integrated strategy holds significant promise for accurate analysis of antibody-drug conjugates, polyclonal antibodies and unknown mAbs including sequences and PTMs, and providing a crucial tool for the discovery and development of therapeutic mAbs.

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Cetuximab 205923-56-4 C107H179N35O36S7 245 suppliers Inquiry
Cetuximab 205923-56-4 C107H179N35O36S7 245 suppliers Inquiry
Cetuximab 205923-56-4 C107H179N35O36S7 245 suppliers Inquiry
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Infliximab 170277-31-3 114 suppliers Inquiry
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