Characterizing Monoclonal Antibody Aggregation Using Charge Detection Mass Spectrometry and Industry Standard Methods.

Abstract

Protein aggregation is a factor in a multitude of neurodegenerative diseases and aggregates of protein-based biotherapeutics can cause toxicity in vivo and adverse patient outcomes. Monoclonal antibody (M)-fluorophore (F) complexes with four different antibody sequences and masses of ∼680 kDa were analyzed using size-exclusion chromatography (SEC) and mass spectrometry using both quadrupole-time-of-flight (QTOF) and charge detection mass spectrometry (CDMS). Higher-order aggregates were not resolved using SEC, but species as large as the MF₂ complex and M₅F₃ were resolved using QTOF and CDMS, respectively. Results from three freeze-thaw cycles and long-term heat stress indicate that both aggregation and degradation occurs. Two of the antibodies form a critical M₂F complex that is sensitive to thermal stress, whereas the other two antibodies undergo degradation and formation of the assembled MF₂ complex in response to freeze-thaw and thermal stressors, respectively. These data show that small differences in mAb sequence can result in significant changes to the aggregation and degradation pathways and highlight the promise of combined mass spectrometry approaches for characterizing how various stress factors affect the stability and aggregation propensity of mAbs.