Understanding the Effects of Site-Specific Light Chain Conjugation on Antibody Structure Using Hydrogen Exchange-Mass...

Area of Science:

• Biochemistry
• Protein Chemistry
• Biotechnology

Background:

• Antibody-drug conjugates (ADCs) are a rapidly advancing class of cancer therapies.
• Site-specific conjugation ensures uniform drug distribution but may affect protein structure and function.

Purpose of the Study:

• To investigate the impact of site-specific conjugation on the higher order structure (HOS) of an antibody drug conjugate (ADC).
• To compare the quality attributes of a site-specifically conjugated ADC with its unconjugated monoclonal antibody (mAb).

Main Methods:

• Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) for thermal stability assessment.
• Size exclusion chromatography (SEC) to evaluate aggregation propensity.
• Differential hydrogen-exchange mass spectrometry (HX-MS) to analyze protein backbone flexibility.

Main Results:

• The ADC exhibited reduced thermal stability in the CH2 transition compared to the unconjugated mAb.
• Conjugation led to earlier aggregation onset and increased aggregation after thermal stress.
• HX-MS data indicated increased flexibility near the conjugation site on the light chain and distal stabilization on the heavy chain.

Conclusions:

• Site-specific conjugation of pyrrolobenzodiazepine to an engineered cysteine residue in the Fab domain alters ADC higher order structure.
• These structural changes may influence ADC safety and efficacy, necessitating careful quality attribute monitoring.