Size Exclusion Chromatography/ Mass Spectrometry Analysis of Antibody Drug Conjugates Using the Agilent 1260 Infinity II Bio-Inert LC

Importance of the Topic

Antibody drug conjugates (ADCs) combine therapeutic monoclonal antibodies (mAbs) with cytotoxic payloads to improve selectivity and reduce off-target toxicity in cancer treatment. Precise drug-to-antibody ratio (DAR) determination and characterization of glycoform heterogeneity are critical for efficacy and safety. Size exclusion chromatography coupled with mass spectrometry (SEC-MS) provides a robust platform for intact and deglycosylated analysis of these biotherapeutics.

Objectives and Study Overview

This study demonstrates the use of Agilent 1260 Infinity II Bio-inert liquid chromatography coupled to quadrupole time-of-flight (Q-TOF) MS for SEC/MS analysis of Trastuzumab (Herceptin) and its ADC form Trastuzumab emtansine (T-DM1, Kadcyla). The goal was to simplify sample preparation, obtain high reproducibility, deconvolute complex mass spectra, and accurately calculate the average DAR.

Methodology and Instrumentation

The workflow involved deglycosylation of mAb and ADC samples using N-glycanase followed by SEC separation in MS-compatible mobile phase (49% water, 50% acetonitrile, 1% formic acid) at 30 °C. Key modules included:

• Agilent 1260 Infinity II Bio-inert Pump, Multisampler, Column Thermostat, and Diode Array Detector
• Agilent 6550 iFunnel Q-TOF Mass Spectrometer in positive ion mode

Main Results and Discussion

SEC-MS analysis of intact Trastuzumab revealed glycoform distributions in the 146–149 kDa range. Deglycosylation simplified spectra, producing a single 145 173 Da peak. For T-DM1, deconvoluted spectra of deglycosylated ADC displayed nine equally spaced peaks, each separated by ~958 Da corresponding to one DM1 payload plus linker. This enabled clear assignment of DAR species from 0 to 8. The average DAR was calculated as 3.1 using Agilent DAR Calculator software. Retention time precision was excellent, with RSD of 0.012% for retention time and 0.486% for peak area.

Benefits and Practical Applications

This SEC-MS approach offers:

• Minimal method optimization and denaturing conditions compatible with labile proteins
• Efficient desalting inline with MS to reduce adduct formation
• Accurate DAR determination for quality control and regulatory submission

Future Trends and Applications

Advancements may include higher resolution MS platforms, integration of ion mobility for conformational analysis, and automated data processing pipelines to accelerate ADC characterization. Expanding this approach to other bioconjugates and biosimilars will support personalized medicine and biosimilar comparability studies.

Conclusion

The Agilent 1260 Infinity II Bio-inert LC-Q-TOF SEC-MS workflow enables robust, high‐resolution characterization of mAbs and ADCs. Deglycosylation simplifies spectra, facilitating easy DAR assignment and ensuring reliable quality control in biopharmaceutical development.

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