Rapid analysis of titer, aggregate and intact mass of antibody therapeutics using multi-dimensional liquid chromatography coupled with native mass spectroscopy

Importance of the Topic

The rapid, accurate characterization of monoclonal antibody therapeutics is critical to ensure safety, efficacy, and quality in biopharmaceutical development. Combining protein A affinity purification with size‐exclusion chromatography and native mass spectrometry in an online two-dimensional liquid chromatography (2D-LC) workflow enables simultaneous measurement of antibody titer, aggregate content, and intact mass, reducing manual handling, turnaround time, and sample loss.

Objectives and Study Overview

• Develop and demonstrate a fast, automated 2D-LC-MS method (<8 min) for multiattribute monitoring of monoclonal antibodies.
• Evaluate performance for purified mAb A and mAb B, and for harvested cell culture supernatant of mAb A.
• Compare glycoform profiles and aggregate levels with conventional approaches.

Methodology and Instrumentation

The workflow uses a heart-cut approach in the first dimension for protein A affinity capture and titer estimation, directly transferring the eluted antibody into a second dimension for size-exclusion chromatography (SEC) coupled to native mass spectrometry. Key steps include:

• Heart-cut transfer from Protein A column to SEC column without manual buffer exchange.
• Native electrospray ionization for intact mass and glycoform analysis.

Instrumentation

• Agilent 1290 Infinity II UHPLC system configured for 2D-LC.
• Bio-Monolith recombinant Protein A column for affinity capture.
• AdvanceBio SEC column (300 Å, 2.7 µm) for size variant separation.
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Agilent JetStream electrospray source.

Key Results and Discussion

Case Study I: Purified mAb A and mAb B

• mAb A: Protein A capture yielded a single peak that SEC-MS resolved into monomer and small high-molecular-weight (HMW) species. Native MS deconvolution identified three major glycoforms (G0F/G0F, G0F/G1F, G1F/G1F) with intact monomer mass ~148 059 Da.
• mAb B: Similar workflow revealed ~22% HMW aggregates and ~78% monomer. Four glycovariants were detected (G0F/G0F to G1F/G2F) with monomer mass ~149 201 Da.

Case Study II: Cell Culture Harvest of mAb A

• Direct analysis of cell culture supernatant showed clear separation of unbound matrix and antibody peak in D1. SEC-MS confirmed ~0.9% aggregates and ~99% monomer, detecting the same three glycoforms as in purified sample.

Benefits and Practical Applications

• Analysis completed in under 8 minutes using 10–15 µg sample, eliminating lengthy buffer exchanges and reducing sample handling risks.
• Simultaneous measurement of titer, aggregate content, intact mass, and glycoform distribution supports quality control and process analytical technology (PAT).
• Improved reproducibility and throughput compared to offline workflows.

Future Trends and Applications

• Extension to additional critical quality attributes such as charge variants and post-translational modifications using alternative chromatographic modalities (e.g., ion exchange, hydrophobic interaction).
• Integration with real-time monitoring in bioreactors and end-to-end continuous manufacturing.
• Adoption of higher-resolution mass analyzers and ion mobility separation for deeper structural insights.

Conclusion

This study demonstrates a robust, fast 2D-LC-MS workflow combining protein A affinity and SEC-MS for comprehensive characterization of monoclonal antibodies. The method offers high throughput, minimal sample preparation, and reliable detection of aggregates and glycoforms, making it well suited for QC and PAT applications in biopharmaceutical development.

References

1. Stoll D.R., Carr P.W. Two-Dimensional Liquid Chromatography: A State of the Art Tutorial. Anal. Chem. 2017;89:519–531.
2. Ehkirch A., et al. A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants. Anal. Chem. 2018;90:13929–13937.
3. Verscheure L., et al. 3D-LC-MS with 2D Multimethod Option for Fully Automated Assessment of Multiple Attributes of Monoclonal Antibodies Directly from Cell Culture Supernatants. Anal. Chem. 2022;94:6502–6511.
4. Sarin D., Kumar S., Rathore A.S. Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS. Anal. Chem. 2022;94(43):15018–15026.