Application of Agilent AdvanceBio Desalting-RP Cartridges for LC/MS Analysis of mAbs

Importance of the Topic

Monoclonal antibodies are a key class of therapeutic proteins and require detailed mass spectrometry characterization. However nonvolatile salts and detergents in sample buffers suppress ionization and degrade MS data quality. Efficient online desalting enhances detection sensitivity and ensures reliable antibody profiling.

Study Objectives and Overview

The study evaluated Agilent AdvanceBio Desalting-RP cartridges for desalting LC separated mAbs prior to MS analysis. One dimensional and two dimensional LC MS configurations were compared with and without the desalting cartridge. The impact of salt concentration on desalting efficiency and method repeatability was also assessed using heart cutting in a 2D LC MS setup.

Methodology and Instrumentation

• One dimensional LC MS: AdvanceBio Desalting-RP cartridge (2.1x12.5mm, 10µm, 1000Å), flow 0.75mL/min, solvents 0.1% formic acid in water and ACN, gradient from 5 to 80%B. MS parameters: gas temperature 350°C, sheath gas 400°C, nebulizer 35 psi, Vcap 5000V, fragmentor 200.
• Two dimensional LC MS: First dimension IEX on Bio MAb NP5 column, gradient of phosphate and NaCl buffers; heart cuts directed to Desalting-RP cartridge as second dimension under reversed phase conditions. Online valve switching permitted inline removal of salts and detergents.
• Instrumentation: Agilent 1290 Infinity II 2D LC, Q TOF MS detector, Agilent AdvanceBio Desalting-RP cartridges, Agilent Bio MAb NP5 IEX column.

Key Results and Discussion

• Comparing 2D LC MS with and without desalting confirmed that cartridges prevented salt induced signal suppression and yielded high quality mass spectra.
• One dimensional analyses of reduced mAbs spiked with up to 300mM NaCl showed consistent glycoform quantitation (G0F, G1F, G2F) despite variations in salt concentration, demonstrating effective desalting.
• Two dimensional heart cut experiments produced clear total ion chromatograms and deconvoluted mass spectra free of salt adducts, confirming online desalting performance.
• Triplicate injections exhibited excellent reproducibility in extracted ion chromatogram profiles and m/z signal intensities, indicating robust injection to injection repeatability.

Benefits and Practical Applications

The AdvanceBio Desalting-RP cartridge enables reliable online salt removal directly in LC MS workflows, improving data quality for therapeutic antibody profiling. Its reproducible performance supports quality control, biotherapeutic development and detailed heterogeneity studies without offline cleanup steps.

Future Trends and Opportunities

Integrating inline desalting cartridges with multidimensional separations and automated platforms can further streamline biomolecule characterization. Future work may extend the approach to other protein classes, improve throughput via miniaturization and apply advanced heart cutting schemes for targeted impurity analysis.

Conclusion

Agilent AdvanceBio Desalting-RP cartridges provide robust online desalting for LC MS analysis of monoclonal antibodies. They effectively remove salts and detergents, prevent ion suppression, and deliver reproducible high quality mass spectra in one and two dimensional setups.

References

Agilent Technologies. Application of Agilent AdvanceBio Desalting-RP Cartridges for LC/MS Analysis of mAbs: A One- and Two-Dimensional LC/MS Study. Application Note 5991-7065EN, 2016.