Antibody-Drug Conjugate (ADC) Drug-to-Antibody Ratio (DAR) Analysis in Serum Using IA-LC-HRMS

Significance of the topic

Antibody-drug conjugates (ADCs) combine the targeting capabilities of monoclonal antibodies with potent cytotoxic drugs. The drug-to-antibody ratio (DAR) critically influences therapeutic efficacy, pharmacokinetics, and safety. Accurate in vivo DAR measurement supports dose optimization and quality control.

Study objectives and overview

This study presents an immunoaffinity ultraperformance liquid chromatography–high resolution mass spectrometry (IA-UPLC-HRMS) workflow to quantify and profile the DAR of ado-trastuzumab emtansine (T-DM1) in mouse serum. Objectives include evaluating method linearity, sensitivity, reproducibility, and stability at pharmacokinetic-relevant concentrations.

Methodology

T-DM1 was captured from serum using streptavidin-coated magnetic beads functionalized with biotinylated HER2 antigen. Elution was achieved with 1% formic acid in 10% acetonitrile. Chromatographic separation employed a reversed-phase ACQUITY UPLC Protein BEH C4 column at 80 °C with a gradient of 0.1% formic acid in water (A) and acetonitrile (B). Detection used a Xevo G2-XS QTof in positive ESI mode with LockSpray calibration. Data acquisition and processing, including deconvolution and automatic DAR calculation, were performed in UNIFI software.

Used instrumentation

• ACQUITY UPLC H-Class PLUS Bio FTN system
• ACQUITY UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1 × 50 mm)
• Xevo G2-XS QTof mass spectrometer
• UNIFI Scientific Information System v1.9.4

Key results and discussion

• Linearity and sensitivity: Robust calibration over 5–100 μg/mL using the 53+ charge state XIC of DAR 3 (m/z 2851.7697); LOQ at 5 μg/mL.
• DAR distribution: Deconvoluted spectra revealed species DAR 0–8 with predominant DAR 3 and 4, including glycoforms (G0F, G1F, G2F).
• Reproducibility: Five replicates in buffer and serum showed low standard deviations in average DAR.
• Stability: In vitro incubation at 37 °C over 120 h yielded negligible changes in signal intensity and DAR.

Benefits and practical applications

• Complements ELISA by providing individual DAR species resolution and structural detail.
• Enables simultaneous qualitative and quantitative assessment of ADC heterogeneity in complex matrices.
• Supports pharmacokinetic and biotransformation studies with high fidelity.

Future trends and applications

Advancements in high-throughput immunoaffinity workflows and enhanced deconvolution algorithms will further streamline DAR profiling. Extending this approach to diverse ADC platforms and integrating pharmacodynamic markers will deepen insights into in vivo ADC behavior. Next-generation HRMS systems may reduce detection limits and accelerate comprehensive analysis of biotherapeutics.

Conclusion

The IA-UPLC-HRMS method offers robust quantification and characterization of T-DM1 DAR in serum, providing critical data on ADC distribution, stability, and heterogeneity. This workflow enhances pharmacokinetic assessments and supports quality control in ADC development.

References

• Mou S, Huang Y, Rosenbaum AI. ADME considerations and bioanalytical strategies for pharmacokinetic assessments of ADCs. Antibodies. 2018;7(41).
• Dere R, Yi JH, Lei C. PK assays for ADCs: case study with ado-trastuzumab emtansine. Bioanalysis. 2013;5(9):1025–1040.
• Kellie JF, Karlinsey MZ. Review of approaches and examples for monitoring biotransformation in protein and peptide therapeutics by MS. Bioanalysis. 2018;10(22):1877–1890.
• Gorovits B, Alley SC, Bilic S, et al. Bioanalysis of ADCs: AAPS ADC Working Group Position Paper. Bioanalysis. 2013;5(9):997–1006.