Online 2D-LC Characterization of Monoclonal Antibodies with Size Exclusion and Weak Cation Exchange Chromatography

Importance of the Topic

The thorough evaluation of monoclonal antibodies for aggregation and charge variants is crucial for ensuring safety and efficacy of biopharmaceuticals. Online two-dimensional liquid chromatography (2D-LC) combines multiple separation mechanisms in a single automated run, significantly improving efficiency and reducing analysis time.

Study Objectives and Overview

This study demonstrates the integration of size exclusion chromatography (SEC) in the first dimension with weak cation exchange chromatography (WCX) in the second dimension for comprehensive mAb characterization. Heart-cutting 2D-LC was used to transfer specific peaks from SEC to WCX, enabling aggregate quantification and charge variant profiling within 13 minutes. High-resolution sampling was applied to detect potential coeluting species and to quantify entire SEC peaks.

Methodology and Instrumentation

A modular Agilent 1290 Infinity II 2D-LC system was configured with a 1260 Infinity Bio-Inert quaternary pump for SEC and a 1290 Infinity II high-speed pump for WCX. A multisampler with cooler, multicolumn thermostat, valve drives with heart-cutting and high-resolution sampling valves, and two diode array detectors were employed. First-dimension separations used an Agilent AdvanceBio SEC 300Å column (4.6×150 mm, 2.7 µm) with 50 mM phosphate buffer (pH 6.2) at 0.5 mL/min. Second-dimension analyses utilized an Agilent Bio MAb column (4.6×50 mm, 1.7 µm) with a phosphate–sodium chloride gradient (25 mM phosphate, pH 6.2 to 500 mM NaCl) at 0.5 mL/min. Data acquisition was managed by Agilent OpenLab CDS ChemStation and 2D-LC software.

Results and Discussion

SEC analysis revealed a monomer peak and a small aggregate peak accounting for approximately 1% of total protein. Heart-cutting WCX resolved two acidic variants and one basic variant alongside the main peak. Precision assessment over seven consecutive injections showed retention time RSDs below 0.08% (1D) and 0.09% (2D), and area RSDs below 0.5% (1D) and 2.1% (2D). High-resolution sampling of eight sequential cuts across the monomer peak detected no coeluting impurities.

Benefits and Practical Applications

The combined 2D-LC approach reduces total run time from traditional 30–40 minutes to 13 minutes, eliminates offline fraction collection, and provides simultaneous quantification of aggregates and charge variants in a single automated workflow. This method enhances laboratory throughput and supports stringent quality control in biopharmaceutical development and manufacturing.

Future Trends and Opportunities

Advancements may include integration of additional chromatographic modes, coupling to mass spectrometry for detailed structural elucidation, and implementation of artificial intelligence for real-time data interpretation. Expanding high-resolution sampling strategies and adopting microflow 2D-LC could further improve sensitivity and reduce solvent consumption.

Conclusion

The online SEC–WCX 2D-LC method provides a rapid, precise, and automated solution for dual attribute analysis of monoclonal antibodies. Its high throughput and reliability make it well suited for routine biopharmaceutical characterization and quality control workflows.

References

1. ICH Q6B. Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.
2. Agilent Technologies. Access Agilent Newsletter, March 2016.
3. Du Y. et al. Chromatographic analysis of acidic and basic species of recombinant monoclonal antibodies. MAbs 2012, 4(5), 578–585.
4. Schneider S. Simple Method Optimization in mAb Charge Variant Analysis using pH Gradients Generated from Buffer Advisor with Online pH and Conductivity Monitoring. Agilent Technologies Application Note 5991-3365EN, 2014.