State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug...

Area of Science:

• Bioconjugation Chemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Antibody-drug conjugates (ADCs) are critical biotherapeutics combining monoclonal antibodies (mAbs) with cytotoxic drugs.
• Traditional ADC production methods yield heterogeneous products due to non-selective conjugation.
• Site-specific conjugation is essential for producing homogeneous ADCs with controlled drug-to-antibody ratios (DAR).

Purpose of the Study:

• To characterize a site-specific DAR2 ADC produced using the GlyCLICK process.
• To evaluate the utility of native mass spectrometry (nMS) and related techniques for monitoring ADC bioconjugation.
• To assess the impact of drug conjugation on antibody stability using ion mobility-based methods.

Main Methods:

• Generation of a site-specific DAR2 ADC via a three-step glycan-based enzymatic remodeling and click chemistry process (GlyCLICK).
• Characterization using state-of-the-art native mass spectrometry (nMS) techniques.
• Monitoring conjugation process and homogeneity using size exclusion chromatography coupled to nMS (SEC-nMS).
• Forced degradation studies and assessment of gas-phase behavior using ion mobility-based collision-induced unfolding (CIU).

Main Results:

• The GlyCLICK process successfully generated a homogeneous site-specific DAR2 ADC.
• SEC-nMS provided direct identification and quantification of reaction products, demonstrating ADC homogeneity.
• SEC-nMS effectively identified fragments during forced degradation studies without drug linker deconjugation.
• CIU revealed increased resistance of the mAb to gas-phase unfolding upon drug conjugation.

Conclusions:

• State-of-the-art nMS methods are powerful tools for investigating drug loading, distribution, and homogeneity in ADCs.
• These methods enable effective monitoring of bioconjugation processes and product evolution.
• nMS and CIU approaches enhance conformational characterization of next-generation mAb-derived products, including engineered nanobodies, bispecific ADCs, and immunocytokines.