Aggregates analysis of a bispecific antibody using Orbitrap Excedion Pro BioPharma Hybrid mass spectrometer with extended mass range

Significance of the Topic

Protein aggregation is a central quality and safety concern in biotherapeutic development. Aggregates can alter efficacy, modify target engagement, provoke immunogenic responses, and complicate pharmacokinetics. Bispecific antibodies (BsAbs) present additional analytical challenges relative to monoclonal antibodies due to asymmetric architectures, multiple domains, and diverse pairing possibilities, making robust aggregate characterization essential for process development, formulation, and regulatory compliance.

Objectives and Study Overview

This study aimed to demonstrate the capability of the Thermo Scientific Orbitrap Excedion Pro BioPharma Hybrid mass spectrometer coupled with size-exclusion chromatography–native mass spectrometry (SEC–nMS) to separate, detect, and identify aggregates and mis‑paired species of a stressed bispecific antibody (intact mass ~103.5 kDa). Two chromatography–MS workflows were compared: an analytical-flow SEC–nMS and a high-flow SEC with low-flow post‑column split to MS, to assess sensitivity, ionization gentleness, and the ability to observe high‑mass species under native conditions.

Methodology

• Sample: A stressed BsAb (intact Mw 103.5 kDa; constituent chains ~52.0 and ~51.5 kDa) supplied by a collaborator.
• Chromatography: Thermo Scientific MAbPac SEC-1 column (5 μm, 4 × 150 mm). Mobile phase: 100 mM ammonium acetate. Column at 30 °C. Standard analytical flow: 180 μL/min, 12 min run.
• High-flow/low-flow approach: identical column and elution conditions but a post-column split using nanoViper Fingertight fittings to deliver ~10 μL/min to the MS, providing gentler ionization and enhanced sensitivity.
• Mass spectrometry: Thermo Scientific Orbitrap Excedion Pro BioPharma Hybrid MS with extended mass range up to m/z 12,000; EASY‑Spray source used for standard work and OptaMax Plus conditions evaluated for ionization gentleness. Acquisition resolutions included 60,000 for analytical-flow experiments and lower effective resolution (3,750) for some high-flow/low-flow acquisitions to compare performance.
• Data processing: Thermo Scientific BioPharma Finder software (v5.4) for deconvolution and glycoform/isoform assignment.

Used Instrumentation

• Orbitrap Excedion Pro BioPharma Hybrid mass spectrometer (extended m/z range to 12,000).
• EASY‑Spray ion source (primary coupling) and OptaMax Plus ion source (evaluated for aggregation stability during ionization).
• Thermo Scientific MAbPac SEC-1 column (5 μm, 4 × 150 mm).
• Thermo Scientific nanoViper Fingertight fittings used for post-column splitting in the high-flow/low-flow workflow.
• Data analysis: Thermo Scientific BioPharma Finder v5.4.

Key Results and Discussion

• Aggregate separation and identification: The stressed BsAb chromatogram separated into five distinct peaks (elution order: tetramer, trimer, dimer, monomer peak 1, monomer peak 2). All species were identified by intact mass after deconvolution.
• Monomer/aggregate abundance: The supplied monomer reference had 99% purity. In the stressed sample, monomer proportion decreased to ~89% (based on UV peak area), with a concomitant increase in aggregates (dimer, trimer, tetramer) and detection of mis‑paired isomers.
• High‑mass detection: The Excedion Pro’s extended m/z capability enabled direct observation of full charge state distributions for very large assemblies — the tetramer charge envelope appeared between m/z ~8,500 and 10,000. Continuous, well-distributed charge state series were observed even for the tetramer.
• Resolution effects: High-resolution acquisition (R = 60,000) improved separation and deconvolution fidelity, enabling clearer observation of N-glycoform distributions on monomer and aggregate species and improved confidence in mis‑pairing assignments. Lower resolution acquisitions (R ≈ 3,750) were still sufficient for overall aggregate detection but lacked the fine glycoform detail.
• Gentleness of ionization and sensitivity: The high‑flow column with low‑flow MS injection approach produced spectra comparable to analytical-flow experiments, indicating that the OptaMax Plus/EASY‑Spray conditions did not induce aggregate dissociation. The low‑flow injection increased sensitivity markedly: for the dimer, one‑eighteenth of the sample amount produced similar MS intensity compared to analytical flow.
• Mis‑paired species and glycoforms: Deconvolution identified mis‑paired isomers (noted as species carrying 2×A2G0F glycoforms), suggesting that the stress conditions promoted mis‑pairing events in addition to noncovalent aggregation.

Benefits and Practical Applications of the Method

• Direct intact-mass measurement: SEC–nMS with an extended mass-range Orbitrap permits unambiguous assignment of aggregate stoichiometry (monomer, dimer, trimer, tetramer) by intact mass rather than only by elution behavior or surrogate detectors.
• Detection of mis‑pairing and heterogeneity: The approach distinguishes mis‑paired isoforms and glycoform variants, valuable for BsAb quality control and mechanistic investigations of stress-induced modifications.
• Flexible workflows: The analytical-flow SEC–nMS enables routine profiling, while the high-flow with low-flow MS injection strategy provides increased sensitivity and gentler ionization for large noncovalent assemblies, supporting both screening and in-depth characterization.
• Compatibility with QC pipelines: The method supports rapid assessment of aggregate levels and species distributions that are relevant for formulation screening, process comparability, and release testing when combined with appropriate validation.

Future Trends and Applications

• Integration with automated sample handling and online buffer exchange to support higher-throughput native MS analysis of complex biologics and membrane proteins.
• Combining extended-range intact MS with middle-up and top-down fragmentation to localize modifications and map noncovalent interaction interfaces within aggregates and mis‑paired species.
• Use in regulatory submissions as orthogonal characterization data supporting aggregate identification and quantitation for complex modalities like BsAbs and multispecific constructs.
• Further optimization of split-flow interfaces and ion source designs to maximize sensitivity for low-abundance aggregates while preserving native assemblies.

Conclusion

The Thermo Scientific Orbitrap Excedion Pro BioPharma Hybrid mass spectrometer, when coupled with SEC–nMS, effectively separates and identifies aggregates and mis‑paired species of a stressed bispecific antibody. The instrument’s extended m/z range allowed observation of full charge envelopes for very large assemblies (tetramer), while the high-flow/low-flow injection strategy provided increased sensitivity without evidence of ionization-induced dissociation. High-resolution acquisition improved glycoform and isoform discrimination. Together, these capabilities make SEC–nMS on an extended-range Orbitrap a powerful tool for BsAb aggregate characterization in research and biopharmaceutical development contexts.

References

• Liu W., Jayasekera H. S., Sanders J. D., Zhang G., Viner R., Marty M. T. Online Buffer Exchange Enables Automated Membrane Protein Analysis by Native Mass Spectrometry. Analytical Chemistry. 2023;95:47.