Conjugation Site Analysis by MS/MS Protein Sequencing

Area of Science:

• Biopharmaceutical Analysis
• Mass Spectrometry
• Protein Chemistry

Background:

• Precise knowledge of drug-linker conjugation sites on monoclonal antibodies (mAbs) is essential for understanding antibody-drug conjugate (ADC) efficacy and safety.
• Current methods may face challenges in analyzing hydrophobic drug-loaded peptides.

Purpose of the Study:

• To develop and present an optimized peptide mapping protocol for accurately determining drug-linker attachment sites on mAbs.
• To enhance the analysis of hydrophobic drug-loaded peptides in ADC characterization.

Main Methods:

• A comprehensive peptide mapping protocol is described, including sample preparation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) setup, and data processing (automated and manual).
• Key improvements focus on maintaining hydrophobic drug-loaded peptides in solution for better chromatographic separation and mass spectrometric detection.

Main Results:

• The protocol enables efficient separation and detection of drug-conjugated peptides.
• Unambiguous identification of drug attachment locations is achieved by utilizing diagnostic fragmentation ions of the payload.
• The method provides a robust approach compared to classical mAb peptide mapping.

Conclusions:

• The described peptide mapping protocol offers an advanced method for precisely locating drug-linker conjugation on mAbs.
• This technique is vital for comprehensive characterization and quality control of antibody-drug conjugates.