Structural Dynamics of a Thermally Stressed Monoclonal Antibody Characterized by Temperature-Dependent H/D Exchange Mass Spectrometry

Differential scanning calorimetry (DSC) is a standard
tool for
probing the resilience of monoclonal antibodies (mAbs) and other protein
therapeutics against thermal degradation. Unfortunately, DSC usually
only provides insights into global unfolding, although sequential
steps are sometimes discernible for multidomain proteins. Temperature-dependent
hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has the potential
to probe heat-induced events at a much greater level of detail. We
recently proposed a strategy to deconvolute temperature-dependent
HDX data into contributions from local dynamics, global unfolding/refolding,
as well as chemical labeling. However, that strategy was validated
only for a small protein (Tajoddin, N. N.; Konermann, L. Anal. Chem. 2020, 92, 10058). The current work explores
the applicability of this HDX framework to the NIST reference mAb
(NISTmAb), a large multidomain protein that is prone to aggregation
and has three melting points. Using global fitting, we were able to
model HDX profiles across the NISTmAb sequence between zero and 95
°C, and for time points between 15 s and 20 min. We uncovered
the enthalpic and entropic contributions of local fluctuations that
govern the conformational dynamics at low temperatures. The CH2 and
CH3 domains were found to be increasingly affected by global unfolding/refolding
in the vicinity of their melting points, although the transiently
unfolded protein displayed significant residual protection. Global
dynamics were not involved in the deuteration of the Fab domains (which
have the highest melting point). Instead, global Fab unfolding was
followed immediately by irreversible aggregation. Our results reveal
that the thermodynamic HDX-MS strategy applied in this work is well
suited for probing spatially resolved dynamics of thermally stressed
large proteins such as mAbs, complementing data obtained by DSC.