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

Area of Science:

• Biochemistry
• Protein Chemistry
• Analytical Chemistry

Background:

• Protein aggregation is implicated in neurodegenerative diseases and can reduce the efficacy and safety of protein-based biotherapeutics.
• Understanding the aggregation and degradation pathways of monoclonal antibodies (mAbs) is crucial for biopharmaceutical development and patient safety.

Purpose of the Study:

• To investigate the impact of different stress conditions on the aggregation and degradation of monoclonal antibody (mAb)-fluorophore (F) complexes.
• To compare the capabilities of size-exclusion chromatography (SEC), quadrupole-time-of-flight (QTOF), and charge detection mass spectrometry (CDMS) in characterizing mAb aggregates.

Main Methods:

• Analysis of monoclonal antibody (M)-fluorophore (F) complexes using size-exclusion chromatography (SEC).
• Characterization of mAb-fluorophore complexes using quadrupole-time-of-flight (QTOF) mass spectrometry.
• Utilizing charge detection mass spectrometry (CDMS) for high-mass aggregate analysis.
• Subjecting complexes to freeze-thaw cycles and long-term heat stress to induce aggregation and degradation.

Main Results:

• Size-exclusion chromatography (SEC) failed to resolve higher-order mAb aggregates.
• QTOF mass spectrometry resolved complexes up to MF₂, while CDMS identified species as large as M₅F₃.
• Two out of four mAbs formed a thermal stress-sensitive M₂F complex.
• The other two mAbs showed degradation and MF₂ complex formation under freeze-thaw and thermal stress, respectively.

Conclusions:

• Monoclonal antibody sequence significantly influences aggregation and degradation pathways under stress.
• Combined mass spectrometry approaches (QTOF and CDMS) are powerful tools for characterizing mAb stability and aggregation.
• Understanding stress-induced changes in mAbs is vital for predicting and preventing adverse patient outcomes.