MSn Analyses for Tryptophan-Conjugated ADC Mimic by Miniature MALDI Digital Ion Trap Mass Spectrometer (MALDI-DIT-MS)

Significance of the topic

The identification of conjugation sites on antibody–drug conjugates (ADCs) is essential for evaluating their structural homogeneity, stability, and therapeutic efficacy. Mapping modification locations at the amino-acid level supports rigorous quality control in biopharmaceutical development.

Objectives and overview

This study produced an ADC mimic by attaching methyl fluorescein-ABNO to tryptophan residues on a humanized IgGκ standard antibody (NISTmAb). It then applied MSn analysis on a compact MALDI digital ion trap mass spectrometer (MALDImini-1) to pinpoint the modification sites.

Methodology and instrumentacion

• Enzymatic digestion: Both modified and unmodified NISTmAb samples were subjected to trypsin digestion followed by C18 ZipTip® desalting.
• MALDI preparation: Peptide mixtures were co-crystallized with 2,5-dihydroxybenzoic acid (DHB) matrix on a stainless-steel target.

Used Instrumentation

MALDImini-1 miniature MALDI digital ion trap mass spectrometer configured for MSn experiments with DHB matrix support.

Main results and discussion

MS analysis revealed four ions exclusive to the modified antibody, notably at m/z 2416.9 and 2560.9. Subsequent MS/MS and MS3 fragmentation produced backbone ions at m/z 1677.7 and 1808.0, matching spectra from the unmodified control. Mascot MS/MS searches identified these peptides as heavy-chain regions 278–291 (FNWYVDGVEVHNAK) and 305–320 (VVSVLTVLHQDWLNGK), confirming conjugation at the tryptophan side chains.

Benefits and practical applications

• Enables rapid, site-specific localization of tryptophan modifications in ADCs without lengthy chromatographic separation.
• Compact system footprint suitable for routine QC and process monitoring in R&D laboratories.
• MSn capability provides detailed structural information essential for regulatory submissions.

Future trends and opportunities

Future workflows may integrate MALDI-DIT-MS with automated sample handling and advanced bioinformatics for high-throughput ADC screening. Extension of this approach to other amino-acid targets or payload chemistries can broaden its applicability in next-generation bioconjugate characterization.

Conclusion

The MALDImini-1 digital ion trap platform demonstrated effective localization of tryptophan-based conjugation sites in an ADC mimic, underscoring its potential as a powerful tool for detailed quality assessment of biotherapeutics.

Reference

[1] Seki Y. et al. J. Am. Chem. Soc. 2016, 138, 10798–10801.