Characterization of Biotherapeutics with High-Resolution Ion Mobility-Mass Spectrometry

Significance of the Topic

Comprehensive characterization of biotherapeutics is crucial to guarantee their safety, efficacy, and consistency in manufacturing and clinical applications. Traditional analytical approaches rely on separate liquid chromatography–mass spectrometry (LC-MS) methods for intact proteins, subunit analysis, peptide mapping, and released glycan profiling. Incorporating high-resolution ion mobility into LC-MS workflows provides an additional separation dimension that enhances the resolution of isoforms, post-translational modifications, and glycan isomers, thereby streamlining quality control and accelerating biopharmaceutical development.

Aims and Overview of the Study

This work demonstrates a unified platform combining liquid chromatography–high-resolution ion mobility–mass spectrometry (LC-HRIM-MS) with Protein Metrics Byos software for HRIM data processing. The goal is to consolidate four key workflows—intact protein analysis, subunit separation, peptide mapping, and released glycan profiling—into a single instrument setup. The study evaluates the performance, reproducibility, and quantitative capabilities of this integrated approach for monoclonal antibody characterization.

Methodology and Instrumentation

Sample Preparation and Workflows:

• Intact Protein: Analysis of a humanized monoclonal antibody standard without prior digestion.
• Subunit Analysis: Reduction of the mAb into light chain (LC) and heavy chain (HC) subunits.
• Peptide Mapping: Denaturation with guanidine HCl, reduction with DTT, alkylation with IAA, tryptic digestion, and desalting for peptide-level PTM identification.
• Released Glycan Analysis: Enzymatic release of N-glycans, labeling with Rapifluor MS reagent, and HILIC separation.

Instrumentation and Software:

• Liquid Chromatography: Agilent 1290 Infinity II UHPLC.
• Columns: PLRP-S for intact/subunit, Waters BEH C18 for peptides, Zorbax HILIC Plus for glycans.
• Mass Spectrometry: Agilent 6545XT Q-TOF system equipped with MOBIE™ HRIM for high-resolution ion mobility separation.
• Data Acquisition and Processing: EyeOn and MassHunter software for acquisition, IM-MS Browser for visualization, Protein Metrics Byos for HRIM workflows including intact, subunit, peptide, and glycan modules.

Main Results and Discussion

Intact and Subunit Analysis:

• The HRIM dimension enabled clear separation of LC and HC subunits, improving deconvolution accuracy and mass precision (mass errors <5 ppm).

Peptide Mapping:

• Mobility separation distinguished modified versus unmodified peptides, enhancing confidence in identifying deamidation, isomerization, oxidation, and glycation sites with reproducible percent modifications across replicates.
• Glycopeptide isomers were resolved in the mobility domain, allowing relative quantitation of site-specific glycosylation variants.

Released Glycan Profiling:

• Distinct mobility features separated released glycan isomers and enabled accurate relative abundance measurements over six replicates with high reproducibility.

Benefits and Practical Applications

• Single-platform approach reduces instrument turnover and method development time for multi-level characterization.
• High-resolution ion mobility adds an orthogonal separation that resolves coeluting species and improves specificity for complex PTMs and glycoforms.
• Streamlined data processing with Protein Metrics Byos accelerates reporting and decision-making in development, QC, and biosimilar comparability studies.

Future Trends and Opportunities

• Integration of ion mobility with emerging fragmentation techniques and higher-field instruments to further enhance structural elucidation.
• Application of the unified LC-HRIM-MS platform to newer biotherapeutic modalities (e.g., antibody-drug conjugates, bispecific antibodies, novel fusion proteins).
• Implementation of machine learning algorithms for automated feature annotation, PTM localization, and isomer classification.
• Expansion into regulated QC workflows, supporting real-time process monitoring and in-line analytics.

Conclusion

The combined LC-HRIM-MS platform with Protein Metrics Byos HRIM workflows delivers a robust, single-instrument solution for in-depth biotherapeutic characterization. By adding high-resolution ion mobility to conventional LC-MS methods, this approach enhances separation of subunits, peptides, and glycans, increases confidence in PTM detection, and streamlines data analysis. The unified workflow can support diverse applications in biologics development, quality control, and biosimilarity assessment.

References

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