Efficient Monoclonal Antibody and Antibody Drug Conjugate Desalting Prior to Mass Spectrometry Using AdvanceBio Desalting-RP Cartridges

Importance of the Topic

The application note presents a streamlined online desalting strategy for monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) using a reversed-phase cartridge prior to electrospray ionization mass spectrometry (ESI-MS). Efficient removal of nonvolatile salts and formulation excipients is essential for high-quality spectral data in biopharmaceutical analysis. The AdvanceBio Desalting-RP cartridge enables rapid desalting with minimal sample handling, improving throughput and data reliability.

Objectives and Study Overview

This study aimed to demonstrate the performance of the AdvanceBio Desalting-RP cartridge for desalting and mass spectrometric analysis of trastuzumab (Herceptin) and its ADC Kadcyla. Key goals included assessing desalting efficiency at varying salt concentrations, reproducibility of peak areas and glycoform percentages, intact antibody detection sensitivity, and applicability across multiple Agilent Q-TOF platforms. Both intact and enzymatically fragmented antibodies were evaluated.

Methodology and Instrumentation

Sample preparation workflows comprised reduction with dithiothreitol, papain or IdeZ digestion, and buffer exchanges. Desalting was performed online on an Agilent 1290 Infinity LC with the AdvanceBio Desalting-RP cartridge (2.1×12.5 mm, 10 µm, 1000 Å). Mobile phases were 0.1% formic acid in water (A) and acetonitrile (B) with a rapid gradient. Flow rate was 400 µL/min, diverting salts to waste before elution. Detection used Agilent 6540, 6550 iFunnel, or 6560 ion mobility Q-TOF MS instruments. Data acquisition and processing employed MassHunter Acquisition, Qualitative Analysis, BioConfirm, and DAR Calculator software.

Key Results and Discussion

Online desalting effectively removed up to 300 mM NaCl, yielding clean total ion chromatograms and deconvoluted spectra for light chain, heavy chain, Fab, Fc, F(ab’)2, and Fc/2 fragments. Peak area precision ranged from 0.5% to 5.7% RSD. Glycoform percentage RSD values were below 3.5% for heavy chain and below 3.4% for fragments. Intact trastuzumab was detected at 50 ng load on the 6550 iFunnel MS. Incorporation of ion mobility separation on the 6560 Q-TOF resolved overlapping charge states in the millisecond timescale. ADC analysis of Kadcyla produced an accurate drug-to-antibody ratio of 3.6 and revealed partial linker-only species in IdeZ-treated fragments.

Benefits and Practical Applications

• Integrates desalting and MS detection in a single run, reducing manual steps.
• Compatible with intact and fragmented antibody workflows.
• Delivers reproducible quantitation of mass variants and glycoforms.
• Supports low-nanogram sensitivity on advanced Q-TOF systems.
• Applicable in QC, R&D, and process analytics for biopharmaceutical development.

Future Trends and Potential Applications

Advancements may include miniaturized or high-throughput desalting formats, deeper integration with next-generation high-resolution MS platforms, and automated workflows for large-scale screening of biologics. The technique is also poised for extension to other protein therapeutics, protein complexes, and real-time monitoring in biomanufacturing.

Conclusion

The Agilent AdvanceBio Desalting-RP cartridge offers a robust and efficient online desalting solution for antibodies and ADCs, enhancing ESI-MS performance by removing interfering salts and excipients. The approach streamlines sample preparation, provides high reproducibility and sensitivity, and supports comprehensive molecular characterization required in biopharmaceutical analysis.

Reference

1. Fenn J B et al. Science 1989, 246, 64-71.
2. Kim M T et al. Bioconjugate Chem 2014, 25, 1223-1232.