Analytical Methods to Monitor Site-Specific ADC Generation with GlyCLICK®
Scientists at the University of Strasbourg and University of Geneva use innovative native MS and IM methodologies for analytical characterization of a site-specific ADC generated with the GlyCLICK technology.
Antibody-drug conjugates (ADCs) combine the benefits of tumor-targeting monoclonal antibodies with the cytotoxic effect of drug payloads covalently linked to the antibody. The ADC generation process has evolved from non-selective and uncontrolled conjugation in early generation products, to site-specific conjugation resulting in homogenous and well-defined ADCs. Conjugation at the antibody Fc glycan sites using the GlyCLICK technology has proven to be an attractive option for the generation of site-specific ADCs.
In this work, the scientists produced a site-specific ADC using the GlyCLICK technology and monitored the conjugation process using an innovative combination of analytical methods. To obtain a homogenous ADC, trastuzumab was site-specifically conjugated with a toxic payload (DM1) using the GlyCLICK technology. Through enzymatic remodeling of the Fc glycans and conjugation using click chemistry, the scientists generated homogenous ADCs carrying two payloads per antibody (DAR 2) using GlyCLICK.
The conjugation process was monitored with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS), allowing for the identification and quantification of all reaction products. The method provided a direct snapshot of the ADC homogeneity and showed further benefits demonstrated in forced degradation studies, where fragments generated upon thermal stress were clearly identified and no deconjugation of the drug linker observed. Using ion mobility-based collision-induced unfolding (CIU) approaches, the authors also assessed the gas-phase behavior of compounds along the conjugation process, with results showing an increased resistance of the mAb against unfolding upon drug conjugation.
In short, this work highlights the GlyCLICK technology for homogenous ADC generation, and the benefits of these innovative approaches to investigate the bioconjugation process for site-specific ADCs.
Deslignière et al., 2021. State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis. Pharmaceuticals. doi: 10.3390/ph14060498
GlyCLICK ADC kit MMAE – Site-specific ADC generation with cleavable linker-payloads carrying MMAE.
GlyCLICK ADC kit PNU – Site-specific ADC generation with cleavable linker-payloads carrying PNU.