ADC Biotransformation Monitoring

SMART Digest IA Streptavidin beads (Part no. 60110-104)

• Antibody-drug conjugates (ADCs) are a promising class of therapeutics

• Assessment of what happens to these molecules in-vivo is a key part of development

• Specifically, assessing the biotransformation of ADCs is key to understanding their stability and pharmacokinetic properties

• The presented method provides a detailed structural analysis of biotransformation products

 Example protocol

• 4ug biotinylated anti-idiotype antibodies added to 30uL of SMART Digest IA Streptavidin beads (Part no. 60110-104)

• Immuno-capture performed by incubating 50 µL human plasma and 30 µL of previously prepared SMART IA beads at 37°C with constant shaking for 2 h.

• After incubation, the bead mixture is washed multiple times.

• SMART Digest buffer and internal standards spiked added to each well

• Sample digested 70°C for 2 h with constant shaking.

• After digestion, 4/5 of the sample volume is cleaned up and concentrated by reversed phase SPE

• (Optional) The remaining 1/5 of the sample volume is incubated overnight at room temperature with papain to release the conjugated payload. Papain digest cleaned up and concentrated by reversed phase SPE

Select Publications

• Yang, J., et al. (2024). Detailed Structural Elucidation of Antibody-Drug Conjugate Biotransformation Species Using High Resolution Multiple Reaction Monitoring Mass Spectrometry with Orthogonal Dissociation Methods. ACS Pharmacology & Translational Science, 8(1), 113–123.

• Huang, Y., et al. (2024). Extensive Biotransformation Profiling of AZD8205, an Anti-B7-H4 Antibody-Drug Conjugate, Elucidates Pathways Underlying Its Stability In Vivo. Analytical chemistry, 96(42), 16525–16533.

• Huang, Y., et al. (2023). Multiplex bioanalytical Methods for comprehensive characterization and quantification of the unique Complementarity-DeTermining-Region deamidation of MEDI7247, an Anti-ASCT2 pyrrolobenzodiazepine Antibody–Drug conjugate. Antibodies, 12(4), 66.

• Yang, J., et al. (2024). Reference‐free thio‐succinimide isomerization characterization by electron‐activated dissociation. Rapid Communications in Mass Spectrometry, 38(23).