A HIGHLY VERSATILE CYCLIC ION MOBILITY – MASS SPECTROMETER FOR ROUTINE TO IN-DEPTH BIOPHARMACEUTICAL CHARACTERIZATION

Importance of the Topic

Monoclonal antibodies are key biotherapeutics with structural heterogeneity arising from production related modifications

Objectives and Study Overview

This work presents a combined approach using subunit analysis, peptide mapping, released glycan profiling and native collision induced unfolding to characterize monoclonal antibodies across cell line development stages

Methodology

• Reduction with dithiothreitol and deglycosylation with PNGase F for subunit analysis
• Denaturation and trypsin LysC digestion for peptide mapping
• Release and RapiFluor MS derivatization of N linked glycans
• Buffer exchange into ammonium acetate prior to native ion mobility CIU

Instrumentation

The SELECT SERIES Cyclic IMS Q TOF mass spectrometer with ion mobility separation and high resolution MS was employed. Data processing used UNIFI for routine analyses and CIUSuite 2 for CIU experiments. Additional instrumentation included HILIC UPLC for glycan separation and micro Bio Spin P 6 columns for buffer exchange

Main Results and Discussion

• Subunit analysis achieved mass resolution of approximately 66 588 for heavy chain charge state and 85 percent sequence coverage by top down MSE
• Peptide mapping with single pass HDMSE provided 95 to 97 percent sequence coverage and enabled collision cross section annotation for modified peptides
• Released glycan profiling with multi pass ion mobility and HILIC separation resolved glycoform isomers and conformational variants
• Native CIU fingerprints for the 24 charge state revealed distinct unfolding transitions among antibody variants with CIU50 analysis

Benefits and Practical Applications

This integrated platform delivers both routine LC MS based identity and glycan profiling and advanced biophysical information such as gas phase structure, conformational stability and unfolding transitions applicable to quality control and comparability studies

Future Trends and Potential Applications

Extending CIU based stability assays under formulation and stress conditions can support formulation screening. Broader application to other protein therapeutics and ligand binding studies may further enhance higher order structure characterization

Conclusion

The SELECT SERIES Cyclic IMS Q TOF platform demonstrates versatility for routine and in depth biopharmaceutical characterization of monoclonal antibodies combining high resolution, coverage and advanced gas phase structural insights

Reference

1. Polasky DA, Dixit SM, Fantin SM, Ruotolo BT. CIUSuite 2 Next Generation Software for the Analysis of Gas Phase Protein Unfolding Data. Analytical Chemistry 2019; DOI 10.1021/acs.analchem.8b05762