Clone Selection Using the Agilent 1290 Infinity Online 2D-LC/MS Solution

Importance of the Topic

Monoclonal antibodies rapidly emerged as key biopharmaceuticals, driving the need for efficient clone screening and quality assurance.
Combining targeted affinity purification with high-resolution mass analysis enables timely selection of cell lines producing correctly folded and modified antibodies.

Objectives and Overview

This study demonstrates an online two-dimensional liquid chromatography–mass spectrometry (2D-LC/MS) workflow for simultaneous enrichment, desalting, quantification, and structural confirmation of monoclonal antibody clones.
A pair of rituximab innovator and biosimilar samples spiked in cell culture medium served as proof-of-principle.

Methodology

The workflow integrates:

• First-dimension affinity capture via Protein A monolithic column, including high-resolution heart-cutting into loop segments.
• Second-dimension reversed-phase desalting and separation on a diphenyl stationary phase directly coupled to MS.
• Time-based valve switching to divert unneeded matrix to waste and channel enriched fractions to the MS.
• Construction of an external calibration curve (0.2–10 µg/µL) using the innovator mAb for accurate concentration determination.

Instrumentation

Equipment and consumables included:

• Agilent 1290 Infinity 2D-LC system with binary and quaternary pumps, dual valve drives, and diode array detectors.
• A multiloop heart-cutting configuration with 40 µL loops for precise sampling.
• Agilent Bio-Monolith Protein A (4.6×12.5 mm) and AdvanceBio RP-mAb Diphenyl (2.1×50 mm) columns.
• Agilent 6530 Accurate-Mass Q-TOF mass spectrometer operating in positive ion mode.
• Data acquisition and analysis via Agilent OpenLab CDS, MassHunter Qualitative Analysis, and BioConfirm software.

Main Results and Discussion

The affinity step achieved selective enrichment of mAbs with minimal matrix interference.
The second-dimension LC delivered narrow peaks and efficient desalting, enabling high-resolution MS detection.
Calibration showed excellent linearity (R² = 0.9996) and recoveries of 98–103 % for spiked samples.
Mass deconvolution revealed distinct glycoform distributions; mirror plots highlighted additional modifications in the biosimilar compared to the innovator clone.

Benefits and Practical Applications

Implementing online 2D-LC/MS accelerates clone screening by combining purification, quantification, and structural characterization in a single automated run.
The approach reduces manual handling, minimizes sample loss, and improves throughput in biopharmaceutical development and QA/QC settings.

Future Trends and Potential Applications

Emerging developments may include faster desalting cartridges, full automation with robotic sample handling, integration of ion-exchange or hydrophilic interaction dimensions, and machine learning-driven data interpretation.
Adaptation to other protein classes and post-translational modification profiling is anticipated.

Conclusion

An online 2D-LC/MS platform based on Agilent technology provides a robust, high-throughput solution for monoclonal antibody clone selection and characterization.
The method offers precise quantification, structural confirmation, and rapid decision-making for biopharmaceutical development.

References

• Scott AM, Wolchok JD, Old LJ. Antibody Therapy of Cancer. Nature Reviews Cancer. 2002;12:278–287.
• Udpa N, Million RP. Monoclonal antibody biosimilars. Nature Reviews Drug Discovery. 2016;15:13–14.
• Gudihal R, Lateef S. Clone selection using a Bio-Monolith Protein A column and LC/MS. Agilent Technologies Application Note. 2014.
• Suresh BCV. LC/MS of intact therapeutic monoclonal antibodies using AdvanceBio RP-mAb. Agilent Technologies Application Note. 2015.
• Suresh BCV, Gudihal R. AdvanceBio Desalting-RP cartridges for online desalting in 2D-LC/MS mAb analysis. Agilent Technologies Application Note. 2016.
• Schneider S. 2D-LC/MS characterization of charge variants using ion exchange and reversed-phase chromatography. Agilent Technologies Application Note. 2016.
• Suresh BCV. Charge heterogeneity analysis of rituximab innovator and biosimilar mAbs. Agilent Technologies Application Note. 2015.