Compliant Monitoring of Monoclonal Antibody Titer and Primary Structure Attributes Using a 2D-LC/MS System with UNIFI Informatics

Importance of the Topic

Two-dimensional liquid chromatography coupled with high-resolution mass spectrometry provides an integrated approach for both quantifying monoclonal antibody (mAb) titer and profiling primary structure attributes such as glycosylation. This combined method addresses the growing need for robust, compliant-ready analytical strategies in biopharmaceutical development and quality control.

Objectives and Study Overview

The study demonstrates how a single chromatographic platform can switch seamlessly between a high-throughput one-dimensional (1D) titer assay and a two-dimensional (2D) workflow for structural confirmation. Using a heart-cut technique, Protein A affinity capture is coupled to reversed-phase LC–MS to measure mAb concentration and verify mass/glycan variants.

Methodology and Instrumentation

• First dimension: Protein A affinity chromatography on a POROS A column, optical detection at 280 nm.
• Second dimension: XBridge Protein BEH C4 column at 80 °C, acetonitrile/formic acid gradient.
• Mass spectrometry: Vion IMS QTof in ESI+ mode, m/z 750–4000, UNIFI software for automated data acquisition and processing.

Key Results and Discussion

• The 1D assay produced a linear calibration curve (R2 = 0.9995) over 0.05–4 mg/mL trastuzumab.
• A 0.1 min heart-cut window efficiently transferred analyte to the second dimension for desalting and mass analysis.
• Deconvoluted spectra revealed major glycoforms (G0F/G0F, G0F/G1F, G1F/G1F, G2F/G2F), enabling confirmation of molecular identity and heterogeneity.

Benefits and Practical Applications

This integrated 1D/2D workflow reduces sample handling, accelerates decision-making during clone selection, process development, and manufacturing monitoring, and maintains regulatory compliance through UNIFI’s audit-ready environment.

Future Trends and Opportunities

Advancements may include multi-heart-cut analyses, deeper structural characterization (e.g., subunit mapping), real-time process analytical technology (PAT) integration, and AI-driven data interpretation to further enhance throughput and insight.

Conclusion

The described 2D-LC/MS system offers a flexible, automated solution for simultaneous mAb quantitation and structural profiling, streamlining workflows while delivering high-quality, compliant-ready data.

Instrumentation Used

• ACQUITY UPLC I-Class PLUS with 2D Technology
• XBridge Protein BEH C4 Column, 300 Å, 3.5 µm
• Vion IMS QTof Mass Spectrometer
• UNIFI Scientific Information System v1.9.4

Reference

• Prentice KM; Wallace A; Eakin CM. Inline Protein A Mass Spectrometry for Characterization of Monoclonal Antibodies. Anal. Chem. 2015, 87, 2023–2028.
• Williams A; Read EK; Agarabi CD; Lute S; Brorson KA. Automated 2D-HPLC Method for Characterization of Protein Aggregation with In-Line Fraction Collection Device. J. Chromatogr. B 2017, 1046, 122–130.
• Sandra K; Steenbeke M; Vandenheede I; Vanhoenacker G; Sandra P. The Versatility of Heart-Cutting and Comprehensive Two-Dimensional Liquid Chromatography in Monoclonal Antibody Clone Selection. J. Chromatogr. A. 2017, 1523, 283–292.