Analysis of Antibody Drug Conjugate Through the Characterization of Drug Antibody Ratios

Importance of the Topic

The precise determination of drug-to-antibody ratio (DAR) is critical for the development and quality control of antibody–drug conjugates (ADCs). DAR directly influences potency, safety, and pharmacokinetics of these targeted therapies. Reliable measurement of DAR supports consistent manufacturing and regulatory compliance in biopharmaceutical research and production.

Objectives and Study Overview

This study demonstrates a robust workflow for measuring DAR in both intact and reduced lysine-conjugated ADCs. Using an Agilent 1290 Infinity Binary LC System coupled to an Agilent 6550 iFunnel Q-TOF, the application note shows how to separate heterogeneous ADC species, deconvolute mass spectra, and calculate DAR values with minimal user intervention.

Methodology

Sample Preparation:

• Reconstitute lyophilized ADC to 5 mg/mL, aliquot and store at –80 °C.
• For intact ADC, dilute to 1 mg/mL in 0.1 % formic acid; optionally deglycosylate with PNGase F at 37 °C overnight.
• For reduced ADC, treat aliquots with 35 mM DTT at 50 °C for 10 min; optional deglycosylation follows reduction.

Chromatography and Mass Spectrometry Conditions:

• Intact ADC on Poroshell 300SB-C8 column, 2.1 × 75 mm, gradient from 15 % to 100 % organic phase.
• Reduced ADC on PLRP-S 1000 Å column, 2.1 × 150 mm, gradient from 20 % to 90 % organic phase.
• Flow rate: 0.4 mL/min; column temperatures: 60 °C (intact) and 85 °C (reduced).
• Mass spectrometer in positive ion mode; dual Jet Stream source; mass ranges: 2 000–5 000 m/z (intact), 800–4 000 m/z (reduced).

Used Instrumentation

• Agilent 1290 Infinity Binary Pump G4220A
• Agilent 1290 Infinity Thermostat Column Compartment G1316C
• Agilent 1290 Infinity Autosampler G4226A
• Agilent 1290 Infinity Multicolumn Thermostat G1330B
• Agilent 6550 iFunnel Q-TOF LC/MS System with Jet Stream source

Key Results and Discussion

Intact ADC:

• Broad chromatographic peaks resolved heterogeneous species.
• Deglycosylation simplified charge envelopes and improved peak resolution.
• DAR values of ~3.6 (glycosylated) and ~3.4 (deglycosylated) were obtained with automated software.

Reduced ADC:

• Light and heavy chains yielded distinct chromatographic profiles (light chain D0–D3, heavy chain D0–D4).
• Deglycosylation of heavy chain simplified spectra, retaining only drug-conjugated peaks.
• Automated DAR calculation reported heavy chain DAR ~1.1, light chain DAR ~0.5, combined DAR ~3.2.

The Agilent MassHunter BioConfirm Software and DAR Calculator efficiently deconvolute spectra, integrate peak areas, and compute weighted DAR values, eliminating manual peak assignment errors.

Benefits and Practical Applications

• High throughput measurement of DAR supports rapid screening during process development.
• Consistent and reproducible results improve batch-to-batch quality control.
• Automated data analysis reduces operator bias and accelerates decision making.

Future Trends and Applications

Emerging needs include integration of high-resolution LC-MS with artificial intelligence for pattern recognition in DAR distributions, real-time monitoring of conjugation reactions, and workflow automation to support large-scale ADC manufacturing. Advances in novel linkers and site-specific conjugation strategies will demand even more accurate DAR measurement tools.

Conclusion

The combination of high-performance LC separation, high-resolution Q-TOF detection, and intuitive data-analysis software provides a reliable platform for DAR characterization in ADCs. This workflow enhances assay precision, throughput, and ease of use, making it suitable for research, development, and QC environments in biopharmaceutical laboratories.

References

• Chari, R. V.; Miller, M. L.; Widdison, W. C. Antibody–drug conjugates: an emerging concept in cancer therapy. Angew. Chem. Int. Ed. Engl. 2014, 53(15), 3796–3827.
• Perez, H. L.; et al. Antibody–drug conjugates: current status and future directions. Drug Discovery Today 2014, 19(7), 869–881.