Complete characterization of a lysine-linked antibody drug conjugate by native LC/MS intact mass analysis and peptide mapping

Significance of the Topic

Antibody drug conjugates combine the specificity of monoclonal antibodies with the potency of cytotoxic small molecules. Complete characterization of such conjugates is critical for ensuring safety, efficacy and consistent quality. Mass spectrometry based approaches provide detailed insight into drug to antibody ratio, glycoform distribution and conjugation site heterogeneity, all of which are essential quality attributes.

Objectives and Study Overview

This study aimed to fully characterize a lysine linked antibody drug conjugate based on trastuzumab emtansine. The objectives were to measure drug to antibody ratio and glycosylation patterns by intact mass analysis under native conditions, and to localize linker drug conjugation sites by peptide mapping. Both workflows were implemented on a single benchtop quadrupole Orbitrap instrument.

Methodology and Instrumentation

Sample Preparation and Chromatography

• Intact Mass Analysis: Online desalting and separation by size exclusion chromatography using aqueous ammonium acetate at physiological pH
• Peptide Mapping: Enzymatic digestion using a fast digest kit followed by reduction and separation by reversed phase chromatography with formic acid and acetonitrile

Instrument Configuration

• Vanquish Binary Pump, Split Sampler FT and Column Compartment for liquid handling
• Q Exactive Plus hybrid quadrupole Orbitrap with BioPharma Option, operated in High Mass Range mode for native intact analysis and Standard mode for peptide mapping
• Data analysis with integrated software using ReSpect deconvolution and peptide mapping workflows

Key Results and Discussion

Native intact mass analysis produced well resolved spectra in a high m/z range, separating charge state envelopes and enabling time resolved deconvolution. A distribution of zero to eight linker drug attachments was observed, with partial chromatographic mobility shifts for higher drug load species. An average drug to antibody ratio of 3.65 was calculated from the main glycoforms. Low level populations of linker only species were also detected, indicating secondary conjugation chemistry. Peptide mapping achieved full sequence coverage and identified thirty of forty four lysine conjugation sites. Conjugated peptides eluted as chromatographic doublets due to stereocenter generation and were confirmed by a signature fragment ion at m/z 547 under higher energy collision dissociation.

Benefits and Practical Applications of the Method

Combining native intact mass with peptide mapping on a single instrument reduces sample handling and avoids deglycosylation or subunit separation steps. The high resolution Orbitrap analyzer provides accurate mass measurements of complex mixtures. Time resolved deconvolution ensures reliable relative abundance estimates for drug load species, supporting precise potency assessment and batch to batch comparability.

Future Trends and Potential Applications

Advances in native mass spectrometry, ion mobility separation and automated data processing will further enhance characterization depth. Emerging linkers and payloads will demand tailored workflows. Increased throughput and integration with artificial intelligence guided analysis promise faster development cycles and real time quality monitoring in biopharmaceutical production.

Conclusion

This work demonstrates that a benchtop quadrupole Orbitrap platform can provide comprehensive characterization of lysine linked antibody drug conjugates. Native intact mass analysis yields accurate drug to antibody ratio and glycoform profiles, while peptide mapping localizes conjugation sites. The combined approach streamlines critical quality attribute assessment and supports robust biotherapeutic development.

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