Analysis of Monoclonal Antibody Digests with the Agilent 1290 Infinity 2D-LC Solution

Significance of the Topic

Biotherapeutic proteins such as monoclonal antibodies exhibit complex structures and post-translational modifications that demand high-resolution analytical techniques. Comprehensive two-dimensional liquid chromatography (2D-LC) provides enhanced peak capacity and orthogonality, enabling detailed peptide mapping for identity, purity, and impurity profiling in drug development and quality control.

Objectives and Study Overview

This application note presents a workflow for dissecting the tryptic digest of trastuzumab using an Agilent 1290 Infinity 2D-LC Solution. Key goals include achieving extensive sequence coverage, assessing method precision, and demonstrating the detection of forced degradation products such as oxidation and deamidation.

Methodology and Instrumentation

The workflow employs:

• First dimension: strong cation exchange (SCX) with a polysulfoethyl column, gradient elution in phosphate buffer with NaCl.
• Second dimension: reversed-phase C18 chromatograph at elevated temperature and fast gradient, modulated every 30 seconds using dual 40 µL loops in co-current mode.
• Detection: diode array detection at 214 nm with high data acquisition rate.

Instrumentation Used

An Agilent 1290 Infinity 2D-LC system was configured with dual binary pumps, autosampler with thermostat, thermostatted column compartment, diode array detector, and a 2-position/4-port valve. Data capture and processing utilized OpenLAB CDS Chemstation with 2D-LC add-on and GC Image LCxLC Edition software.

Results and Discussion

Contour plots revealed over 100 tryptic peptides distributed across both dimensions, confirming strong orthogonality and comprehensive sequence coverage. Precision tests (n=5) on selected peptides yielded RSDs for peak volume below 5% and retention time variations under 0.3% in the second dimension. Forced degradation studies clearly differentiated native peptides from oxidative and pH-induced degradation products: an oxidation of methionine in peptide T41 and deamidation of asparagine in peptide T46 were unambiguously detected despite co-elution challenges in single-dimension methods.

Benefits and Practical Applications

• High resolution and orthogonality support detailed identity and purity assessments.
• Excellent method precision enables robust batch-to-batch and originator-biosimilar comparisons.
• Forced degradation profiling facilitates impurity detection and stability studies.

Future Trends and Opportunities

Advances in column chemistries and faster modulation technologies will further increase peak capacity and throughput. Integration with high-resolution mass spectrometry and automated data processing will streamline biopharmaceutical characterization and support real-time release testing in regulated environments.

Conclusion

The Agilent 1290 Infinity 2D-LC Solution provides a powerful, precise, and reproducible platform for peptide mapping of monoclonal antibodies. Its strong orthogonality and sensitivity to modifications make it ideal for identity verification, impurity profiling, and comparative biosimilarity studies.

References

1. K. Sandra et al., The power of liquid chromatography-mass spectrometry in the characterization of protein biopharmaceuticals, LCGC Europe Supplement, May 2013.
2. I. Francois et al., Comprehensive liquid chromatography: fundamental aspects and practical considerations – a review, Anal. Chim. Acta, 2009, 64, 14–31.
3. K. Sandra et al., Highly efficient peptide separations in proteomics. Part 2: bi- and multidimensional liquid-based separation techniques, J. Chromatogr. B, 2009, 877, 1019–1039.