Rapid monitoring of mAb aggregates during the purification process development of therapeutic mAbs using a modified Orbitrap hybrid MS

Significance of the Topic

Monoclonal antibodies (mAbs) are critical biotherapeutics, yet protein aggregation during production poses risks to efficacy and safety. Rapid, reliable monitoring of aggregates is essential for downstream purification process development and quality control. Native intact mass analysis combined with size-exclusion chromatography (SEC) and UV detection enables direct molecular weight measurement of aggregates, supporting real-time decision-making in mAb manufacturing.

Study Objectives and Overview

The primary aim was to establish an SEC-UV-MS platform for:

• Accurate determination of molecular weights for monomers and high-mass species (HMMS) of mAbs.
• Quantitative monitoring of aggregate levels during purification.
• Evaluation of POROS Caprylate mixed-mode cation exchange resin effectiveness in removing aggregates.

The workflow was applied to an in-house mAb sample (mAb1) before and after polish purification.

Materials, Methods, and Instrumentation

Sample Preparation:

• NISTmAb standard and tetrameric β-galactosidase (≈466 kDa) for sensitivity and accuracy tests.
• In-house mAb1 samples collected from Protein A pool (high aggregates) and post-polish pool.

Chromatography and Detection:

• Vanquish Horizon UHPLC system with MAbPac SEC-1 column (4 × 300 mm, 300 Å) at 30 °C, 250 µL/min, 50 mM ammonium acetate buffer.
• UV detection at 280 nm.

Mass Spectrometry:

• Orbitrap Excedion Pro BioPharma hybrid mass spectrometer in full MS mode.
• Extended high mass range up to m/z 12,000; resolution 60,000 at m/z 200.
• ESI source and MS parameters optimized for high-mass protein analysis.

Data Analysis:

• BioPharma Finder 5.3 software for deconvolution and average mass determination.

Results and Discussion

Sensitivity and Mass Accuracy:

• β-galactosidase tetramer measured at 466,560 Da, matching the expected 466,310 Da within <100 ppm.
• NISTmAb deconvoluted glycoforms achieved <10 ppm mass accuracy.

Aggregate Monitoring:

• SEC-UV profiles showed a 2.2% HMMS peak in pre-polish sample, absent post-polish.
• Deconvolution of HMMS peaks identified mAb1 plus two light chains (≈192,605 Da) and intact mAb1 dimer (≈294,159 Da).

The data confirm the platform’s capacity to detect and quantify mAb aggregates directly and to assess resin performance.

Benefits and Practical Applications

This method offers:

• Label-free, direct molecular weight measurement of monomers and aggregates.
• High sensitivity for detecting low-level HMMS in early development.
• Quantitative monitoring to guide process optimization and resin selection.
• Compatibility with established UHPLC-SEC workflows.

Future Trends and Applications

Anticipated developments include:

• Expansion of mass range and resolution for multi-subunit complexes above 500 kDa.
• Integration with automated inline sampling and feedback control for continuous processing.
• Advanced algorithms and AI-driven deconvolution to streamline data interpretation.
• Coupling with orthogonal separations (e.g., ion exchange) for comprehensive process analytics.

Conclusion

The developed SEC-UV-native intact mass platform leveraging the Orbitrap Excedion Pro BioPharma MS enables rapid, accurate detection and quantification of mAb aggregates. It provides a robust tool for process development, ensuring control of critical quality attributes and supporting efficient purification strategies.

References

1. McGee JP et al. Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry. Analytical Chemistry. 2021;93(5):2723–2727.