A Quick and Efficient Sample Cleanup for Biomolecule Analysis

Significance of the Topic

Effective sample cleanup is critical in biopharmaceutical analysis to remove excipients, preservatives, salts, and detergents that can compromise chromatographic columns, contaminate mass spectrometry detectors, and obscure accurate protein characterization.

Objectives and Overview of the Study

This application note demonstrates a rapid, user-friendly approach for buffer exchange and desalting of formulated monoclonal antibodies using centrifugation with gel filtration cartridges. The goal is to simplify sample preparation for intact protein and released glycan analysis by LC/MS, ensuring minimal sample loss and compatible buffer conditions for downstream workflows.

Methodology and Instrumentation

Sample cleanup was performed using Agilent AdvanceBio Spin cartridges and 96-well plates via stepwise centrifugation to displace storage solution and exchange into desired buffers. Intact rituximab in histidine buffer (12.5 mM, pH 6.5) was processed to remove polysorbate 80 and salts. For glycan analysis, samples underwent denaturation, enzymatic deglycosylation, and labeling with InstantPC before hydrophilic interaction liquid chromatography (HILIC) separation.

Main Results and Discussion

Desalting tests showed efficient removal of 0.8 M NaCl, as measured by conductivity. LC/MS of untreated rituximab revealed early eluting hydrophilic impurities and a complex polysorbate 80 pattern. Following spin-column cleanup, these components were nearly eliminated while the intact antibody peak remained unchanged. Deconvolution of MS spectra identified major glycoforms (G0F, G1F, G2F) consistent with expectations. Released glycan analysis confirmed glycan distribution and demonstrated that buffer exchange prevented histidine interference with InstantPC labeling.

Benefits and Practical Applications of the Method

• Rapid cleanup in under 10 minutes per sample
• High recovery with minimal concentration change
• Compatibility with downstream LC/MS workflows
• Prevents column fouling and detector contamination

Future Trends and Potential Applications

Integration with automated, high-throughput platforms could further accelerate sample preparation. Advances in spin-column chemistries may expand applicability to a broader range of biomolecules, complex formulations, and multi-dimensional separations.

Conclusion

Centrifugation with gel filtration cartridges offers a straightforward, efficient solution for buffer exchange and desalting of monoclonal antibodies. This approach enhances data quality in intact protein and glycan analyses while saving time and preserving sample integrity.

Used Instrumentation

• Agilent 1290 Infinity II Bio LC system
• Agilent PLRP-S 1000 Å reversed-phase column (2.1×50 mm, 5 µm)
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer
• AdvanceBio Spin columns and 96-well plates
• AdvanceBio Gly-X InstantPC deglycosylation and labeling kit
• Hydrophilic interaction LC column (AdvanceBio Amide HILIC, 2.1×150 mm, 1.8 µm)
• Standard laboratory centrifuge and pipettes