posted on 2021-08-27, 00:05authored byEvolène Deslignière, Thomas Botzanowski, Hélène Diemer, Dale A. Cooper-Shepherd, Elsa Wagner-Rousset, Olivier Colas, Guillaume Béchade, Kevin Giles, Oscar Hernandez-Alba, Alain Beck, Sarah Cianférani
Monoclonal antibodies
(mAbs) have taken on an increasing importance
for the treatment of various diseases, including cancers and immunological
disorders. Disulfide bonds play a pivotal role in therapeutic antibody
structure and activity relationships. Disulfide connectivity and cysteine-related
variants are considered as critical quality attributes that must be
monitored during mAb manufacturing and storage, as non-native disulfide
bridges and aggregates might be responsible for loss of biological
function and immunogenicity. The presence of cysteine residues in
the complementarity-determining regions (CDRs) is rare in human antibodies
but may be critical for the antigen-binding or deleterious for therapeutic
antibody development. Consequently, in-depth characterization of their
disulfide network is a prerequisite for mAb developability assessment.
Mass spectrometry (MS) techniques represent powerful tools for accurate
identification of disulfide connectivity. We report here on the MS-based
characterization of an IgG4 comprising two additional cysteine residues
in the CDR of its light chain. Classical bottom-up approaches after
trypsin digestion first allowed identification of a dipeptide containing
two disulfide bridges. To further investigate the conformational heterogeneity
of the disulfide-bridged dipeptide, we performed ion mobility spectrometry–mass
spectrometry (IMS–MS) experiments. Our results highlight benefits
of high resolution IMS–MS to tackle the conformational landscape
of disulfide peptides generated after trypsin digestion of a humanized
IgG4 mAb under development. By comparing arrival time distributions
of the mAb-collected and synthetic peptides, cyclic IMS afforded unambiguous
assessment of disulfide bonds. In addition to classical peptide mapping,
qualitative high-resolution IMS–MS can be of great interest
to identify disulfide bonds within therapeutic mAbs.