Automated Biotransformation Analysis of ADCs using FabRICATOR

The development of antibody-drug conjugates (ADCs) has evolved from first generation formats prepared by random conjugation technologies to next generation ADCs generated by site-specific conjugation. While significant improvements in overall efficacy and safety is displayed by site-specific formats, bioanalysis remains challenging due to complex in vivo biotransformation events including deconjugation, linker-payload cleavage and payload metabolism.

In this work, scientists at Bristol-Myers Squibb describe the development of an automated and fast affinity capture method using a cartridge-based platform combined with LC-HRMS analysis for biotransformation assessment of site-specific ADCs.

The ADCs were captured on streptavidin cartridges coated with a biotinylated anti-human capture reagent followed by on-cartridge digestion of the immobilized conjugates prior to elution and LC-HRMS analysis. Enzymatic digestion was performed based on the site of conjugation to reduce sample complexity by Fc region or N-glycan removal. The FabRICATOR enzyme was used for site-specific digestion below the hinge to generate homogenous F(ab’)2 fragments from ADCs carrying Fab conjugated payloads, while PNGase F was used for deglycosylation of Fc conjugated ADCs.

The optimized on-cartridge digestion step was performed in only 15 min with a total sample preparation time of less than 4 hours. By coupling on-cartridge digestion and HRMS analysis the scientists were able to accurately identify ADC catabolites with very low mass shifts without generating 25 kDa fragments by interchain disulfide reduction along with monitoring changes in payload distribution. In vitro and in vivo biotransformations of multiple ADCs including changes in the DAR distribution were evaluated using the new method that has a broad application and can potentially be used for other complex biologics including tethered antibodies, fusion proteins, probodies, and bispecifics.

Jashnani et al., 2021. Automated and Faster Affinity Capture Method for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates. Analytical Chemistry. doi: https://doi.org/10.1016/j.aca.2021.338347

FabRICATOR – Cystine protease for digestion of mAbs, ADCs and Fc-fusion proteins at a specific site below the hinge.