Quantitative collision-induced unfolding differentiates model antibody-drug conjugates

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Antibody-drug conjugates (ADCs) are effective cancer therapeutics, comprising antibodies linked to potent drugs.
• Conjugation at lysine residues can introduce structural heterogeneity, impacting antibody conformation.
• Characterizing these structural changes is crucial for ADC development.

Purpose of the Study:

• To develop and apply a workflow for structural characterization of lysine-linked antibody-biotin conjugates.
• To compare the sensitivity of native ion mobility-mass spectrometry (IM-MS) and collision induced unfolding (CIU) in detecting conjugation-induced conformational changes.
• To investigate the correlation between CIU and differential scanning calorimetry (DSC) for stability assessment.

Main Methods:

• Native ion mobility-mass spectrometry (IM-MS) for structural analysis.
• Gas-phase unfolding (CIU) to probe conformational stability.
• Liquid Chromatography-Mass Spectrometry (LC-MS) to determine conjugation states.
• Size exclusion chromatography (SEC) for comparing hydrodynamic properties.

Main Results:

• Native IM-MS and SEC were insufficient to resolve subtle conformational changes in antibody-biotin conjugates.
• CIU successfully detected structural and stability differences upon biotin conjugation, outperforming native IM-MS.
• CIU and DSC data indicated destabilization of antibody-biotin conjugates, suggesting a correlation between the methods.

Conclusions:

• CIU offers enhanced sensitivity for characterizing antibody-drug conjugates compared to native IM-MS alone.
• The findings highlight the utility of IM-MS and CIU in the development of antibody-drug conjugates.
• A potential correlation between CIU and DSC for stability assessment in ADCs was identified.