Glycosylated and Deglycosylated Subunit Analysis of Antibody Drug Conjugates

Significance of the Topic

Antibody–drug conjugates (ADCs) represent a critical class of targeted biotherapeutics that combine the specificity of monoclonal antibodies with potent cytotoxic agents. Detailed characterization of ADC subunits, encompassing both glycosylated and deglycosylated forms, is essential to ensure consistent drug–antibody ratios (DAR) and to maintain safety and efficacy in preclinical and clinical development.

Objectives and Study Overview

This study presents a streamlined workflow for subunit analysis of trastuzumab emtansine (T-DM1) using benchtop QTOF mass spectrometry. Key objectives include:

• Developing a robust method to independently assess light and heavy chain drug loading
• Comparing glycosylated and deglycosylated subunit profiles
• Calculating DAR values with BioPharmaView software

Experimental Methodology and Instrumentation

Sample Preparation:

• Deglycosylation: PNGase F treatment following standard protocol
• Reduction: TCEP addition and incubation at 56 °C for 30 minutes

Chromatographic Conditions:

• Column: Agilent Poroshell 300SB-C8, 1.0 × 75 mm, 5 µm
• Mobile phase A: 0.1% formic acid in water
• Mobile phase B: 0.1% formic acid in acetonitrile
• Flow rate: 0.2 mL/min; Column temperature: 75 °C; Run time: 10 min

Mass Spectrometry Conditions:

• Instrument: SCIEX X500B QTOF
• Source: Curtain gas 30, GS1/GS2 50, Ion spray voltage 5000 V, Source temp 400 °C
• Acquisition: m/z 900–4000, Declustering potential 250 V, Accumulation 0.5 s, Time bins 80
• Intact Protein Mode and Large Proteins (>70 kDa) enabled

Main Results and Discussion

Glycosylated Subunit Analysis:

• Light chain: 55% unmodified, 32% with one drug, 13% with two drugs; up to two drug moieties detected
• Heavy chain: 34% unmodified, 35% with one drug, 22% with two, 9% with three; up to three drug moieties detected
• Calculated DAR: light chain 0.59; heavy chain 1.07
• Minor species (+221 Da) corresponding to linker modification without payload observed at low intensity

Deglycosylated Subunit Analysis:

• Subunit profiles closely matched glycosylated forms
• Calculated DAR: light chain 0.52; heavy chain 1.01
• Minor +221 Da species confirmed

Benefits and Practical Applications

This approach offers:

• Rapid and straightforward subunit-level ADC characterization
• Direct DAR calculation without extensive sample manipulation
• Compatibility with batch processing for quality control and comparability studies

Future Trends and Possibilities

Advancements may include:

• Automated workflows integrating sample prep to data reporting
• High-resolution MS tools for deeper glycoform and positional analysis
• Application to diverse ADC formats and novel payload chemistries

Conclusion

The described benchtop QTOF method enables efficient analysis of both glycosylated and deglycosylated ADC subunits, providing accurate DAR measurements essential for ADC development and quality assessment.

References

1. Kim MT et al. Bioconjugate Chem. 2014;25:1223–1232.
2. Chen Y et al. Bioconjugate Chem. 2016;27:2037–2047.
3. Chen L et al. mAbs. 2016;8(7):1210–1223.