Intact Analysis of Antibody Drug Conjugates

Importance of the topic

Antibody–drug conjugates (ADCs) represent a rapidly growing class of targeted biotherapeutics that combine the selectivity of monoclonal antibodies with potent cytotoxic drugs. Precise determination of the drug-to-antibody ratio (DAR) is essential for ensuring consistent safety and efficacy profiles during development and manufacturing.

Objectives and study overview

This work aims to demonstrate a streamlined workflow for intact ADC analysis using a compact benchtop X500B QTOF mass spectrometer. Trastuzumab emtansine (T-DM1) was evaluated in both its native glycosylated form and after enzymatic deglycosylation to assess DAR and glycoprofiles efficiently.

Methodology

• Sample preparation: T-DM1 analyzed either directly or following N-glycan removal with PNGase F.
• LC conditions: Reversed-phase chromatography on a Poroshell C8 column at 75 °C with 0.1 % formic acid in water and acetonitrile; flow rate 0.2 mL/min.
• MS acquisition: X500B QTOF in intact protein mode, mass range 900–4000 m/z, ion spray 5000 V, source temp 400 °C.
• Data processing: BioPharmaView software for spectral reconstruction and DAR calculation.

Used instrumentation

• Benchtop X500B QTOF Mass Spectrometer with SCIEX OS.
• Exion LC System with Agilent Poroshell 300SB-C8 column (1.0×75 mm, 5 µm).
• BioPharmaView 2.0.1 for peak reconstruction and DAR computation.

Main results and discussion

• Glycosylated T-DM1 raw spectra displayed high complexity due to overlapping glycoforms and multiple drug attachments. Reconstruction resolved species carrying 0 to 8 drugs, with consistent glycoprofiles across DAR species.
• Observation of a 221 Da mass shift indicated a crosslinked linker intermediate formed by proximal lysine reaction, confirming literature reports.
• Calculated DAR for glycosylated T-DM1 was 3.49, closely matching the literature value of approximately 3.5.
• Deglycosylation simplified the spectrum by removing glycan heterogeneity. Reconstructed data still showed the 221 Da shift. DAR for deglycosylated T-DM1 was determined as 3.26.

Benefits and practical applications

• High-resolution data from a benchtop QTOF enables routine characterization of complex biologics like ADCs.
• Efficient workflow combining rapid LC separation and automated software reconstruction accelerates DAR determination.
• Capability to detect minor modifications, such as linker crosslinks, enhances product quality control.

Future trends and potential applications

• Integration of automated sample preparation and data analysis for higher throughput in ADC screening.
• Advancements in software algorithms for deeper structural elucidation of post-translational modifications.
• Expansion of benchtop high-resolution MS platforms to include multi-attribute monitoring in biopharma QC environments.

Conclusion

The benchtop X500B QTOF platform, paired with BioPharmaView software, provides a robust solution for intact ADC analysis. It delivers high mass accuracy and resolution necessary for clear identification of glycoforms, drug load distributions, and linker variants, supporting accelerated ADC development and quality assessment.

References

1. Michael T Kim, Yan Chen, Joseph Marhoul and Fred Jacobson Statistical Modeling of the Drug Load Distribution on Trastuzumab Emtansine Kadcyla Bioconjugate Chem 2014 25 1223–1232
2. Yan Chen, Michael T Kim, Laura Zheng, Galahad Deperalta and Fred Jacobson Structural Characterization of Cross-Linked Species in Trastuzumab Emtansine Kadcyla Bioconjugate Chem 2016 27 2037–2047
3. Liuxi Chen, Lan Wang, Henry Shion, Chuanfei Yu, Ying Qing Yu, Lei Zhu, Meng Li, Weibin Chen and Kai Gao In-depth structural characterization of Kadcyla ado-trastuzumab emtansine and its biosimilar candidate MABS 2016 8 7 1210–1223