Aggregation of protein-based therapeutics can occur during
development,
production, or storage and can lead to loss of efficacy and potential
toxicity. Native mass spectrometry of a covalently linked pentameric
monoclonal antibody complex with a mass of ∼800 kDa reveals
several distinct conformations, smaller complexes, and abundant higher-order
aggregates of the pentameric species. Charge detection mass spectrometry
(CDMS) reveals individual oligomers up to the pentamer mAb trimer
(15 individual mAb molecules; ∼2.4 MDa) whereas intermediate
aggregates composed of 6–9 mAb molecules and aggregates larger
than the pentameric dimer (1.6 MDa) were not detected/resolved by
standard mass spectrometry, size exclusion chromatography (SEC), capillary
electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole
time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC,
and CE-SDS did not resolve partially or more fully unfolded conformations
of each oligomer that were readily identified using CDMS by their
significantly higher extents of charging. Trends in the charge-state
distributions of individual oligomers provides detailed insight into
how the structures of compact and elongated mAb aggregates change
as a function of aggregate size. These results demonstrate the advantages
of CDMS for obtaining accurate masses and information about the conformations
of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability
to investigate structural changes that occur in small, soluble oligomers
during the earliest stages of aggregation for antibodies or other
proteins.