A streamlined drug-to-antibody ratio determination workflow for intact and deglycosylated antibody-drug conjugates

Importance of the topic

Antibody drug conjugates represent a rapidly growing class of targeted therapeutics. The ratio of cytotoxic payload to antibody, known as the drug to antibody ratio or DAR, is a critical quality attribute influencing efficacy and safety. Accurate monitoring of DAR distribution in biological samples supports informed decision making in bioconjugate development and pharmacokinetic studies.

Objectives and Study Overview

This study demonstrates a fully automated workflow for determination of DAR in intact and deglycosylated antibody drug conjugates spiked into serum. The process integrates antigen immobilization, affinity capture, on cartridge deglycosylation, and LC MS analysis to streamline sample preparation and improve reproducibility.

Methodology and Used Instrumentation

A biotinylated extracellular domain of HER2 was immobilized on streptavidin cartridges using an Agilent AssayMAP Bravo platform. Samples of trastuzumab emtansine (T DM1) in rat serum were diluted and passed through the cartridges for affinity purification. On cartridge deglycosylation was performed with PNGase F at 37 °C to simplify mass spectra. Eluted antibodies were analyzed by LC MS using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6545XT AdvanceBio quadrupole time of flight mass spectrometer.

Main Results and Discussion

• Recovery and purity of T DM1 following automated affinity capture exceeded expectations as judged by total ion chromatograms.
• On cartridge deglycosylation removed glycoforms and enhanced signal intensity without compromising antibody integrity.
• Extracted ion chromatogram based quantification showed coefficients of variation below 10 percent for most tested concentrations, demonstrating high reproducibility.
• A linear response was observed for on column loads from 3.125 to 100 nanograms of T DM1, with R squared values above 0.998.
• Average DAR values were consistently measured at approximately 3.5 across a range of sample loads, matching expected values and establishing a limit of quantification at 0.3125 micrograms per milliliter in serum.

Benefits and Practical Applications

This automated workflow reduces manual handling, limits potential errors, and scales to 96 samples per run. The streamlined process decreases hands on time to less than five hours from sample to result. It supports high throughput DAR determination for development or quality control of antibody drug conjugates in complex biological matrices.

Future Trends and Potential Uses

Future enhancements may include integration of ion mobility for improved separation of proteoforms, coupling with advanced data analysis platforms for real time DAR monitoring, and adaptation to other conjugate formats. Expanding the range of capture ligands and enzymatic treatments on cartridge will broaden applicability to diverse biotherapeutics.

Conclusion

An end to end automated workflow using Agilent AssayMAP Bravo and high resolution LC MS enables reliable and scalable determination of drug to antibody ratio in serum samples. The approach offers high reproducibility, minimal hands on time, and accurate DAR measurement for both intact and deglycosylated antibody drug conjugates.

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