Quantification of the Antibody Drug Conjugate, Trastuzumab Emtansine, and the Monocolonal Antibody, Trastuzumab, in Plasma Using a Generic Kit-Based Approach

Importance of the Topic

Monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) have become critical therapeutic agents due to their target specificity and potency while minimizing systemic toxicity. Robust quantification of these biotherapeutics in biological matrices is essential for pharmacokinetic, pharmacodynamic, and bioequivalence studies. Traditional ligand binding assays exhibit limitations in dynamic range, specificity, and reagent variability, driving the adoption of LC–MS methods. The development of streamlined, kit-based workflows addresses challenges in method complexity and reproducibility, enabling broader application of LC–MS in drug development.

Objectives and Study Overview

This study aimed to establish a standardized, generic kit-based approach for the accurate quantification of trastuzumab and its ADC derivative ado-trastuzumab emtansine (T-DM1) in rat plasma. Using the ProteinWorks eXpress Direct Digest Kit and a five-step digestion protocol, the work evaluated assay sensitivity, linearity, precision, and accuracy over a wide concentration range.

Methodology and Protocol

Plasma samples spiked with trastuzumab or T-DM1 (0.1–500 µg/mL) were directly digested (35 µL sample) following reduction and alkylation steps. A generic murine IgG served as internal standard. Peptide separation was performed on an ACQUITY UPLC Peptide BEH C18 column (2.1×150 mm, 1.7 µm) with a gradient elution (0.1% formic acid in water/acetonitrile) at 0.3 mL/min and column temperature 55 °C. Signature tryptic peptides (IYPTNGYTR, FTISADTSK, GPSVFPLAPSSK) were monitored by a Xevo TQ-S triple quadrupole in MRM mode.

Used Instrumentation

• ACQUITY UPLC system
• ACQUITY UPLC Peptide BEH C18, 300 Å, 1.7 µm, 2.1 × 150 mm column
• Xevo TQ-S mass spectrometer with ESI+
• MassLynx v4.1 software

Main Results and Discussion

The assay demonstrated linearity over 0.5–500 µg/mL with r2 >0.99 and lower limits of quantification of 0.5–1 µg/mL. Accuracy and precision met validation criteria (CV <8%, accuracy 90–110%). The lysine-free peptide IYPTNGYTR provided reliable quantification for both mAb and ADC due to absence of miscleavage issues. Lysine-containing peptides exhibited potential underestimation when drug loads blocked cleavage, illustrating the importance of peptide selection. Chromatograms confirmed stable retention and sensitive detection of signature peptides. Conjugated miscleavage peptides (FTISADTSKNTAYLQMNSLR, GPSVFPLAPSSKSTSGGTAALGCLVK) were detected and increased proportionally with T-DM1 concentration, enabling direct monitoring of payload occupancy.

Benefits and Practical Applications

• Eliminates extensive method development via kit-based reagents and protocol
• Supports multiplexed quantification of mAbs and ADCs
• Extends dynamic range suitable for pharmacokinetic studies
• Reduces assay variability with premeasured reagents

Future Trends and Potential Applications

Emerging workflows will integrate higher-throughput digestion platforms and advanced data analysis to support large-scale ADC drug screening. The approach can be adapted to diverse mAb–payload combinations and incorporated into automated bioanalytical pipelines. Multiplex quantification of multiple therapeutic antibodies in a single run and deeper characterization of ADC catabolites represent promising extensions.

Conclusions

The described generic kit-based LC–MS/MS workflow provides a simple, reproducible, and sensitive method for quantifying trastuzumab and T-DM1 in plasma. By leveraging streamlined digestion and robust MRM assays, the approach addresses key limitations of traditional immunoassays and facilitates timely decision-making in bioanalytical studies.

References

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3. DrugBank. Trastuzumab emtansine. Accessed online.
4. FDA Guidance for Industry: Bioanalytical Method Validation. CDER.