Agilent 1290 Infinity II 2D-LC solution Biopharmaceutical Polymer Analysis

Importance of the topic

Two-dimensional liquid chromatography (2D-LC) addresses the growing need for higher resolving power in the analysis of complex biopharmaceutical samples. By coupling two orthogonal separation dimensions, 2D-LC enhances peak capacity, improves removal of interfering matrix components and accelerates sample throughput, making it indispensable for peptide mapping, charge variant profiling, aggregation analysis and glycan characterization.

Objectives and overview of the application note

This application note details the Agilent 1290 Infinity II 2D-LC Solution designed for biopharmaceutical polymer analysis. It introduces system configuration, operational modes, dedicated bio-inert components and integrated software. Four case studies illustrate the performance benefits in monoclonal antibody and glycan analysis, highlighting automated workflows that combine multiple quality-attribute assays.

Methodology and workflows

Principles of two-dimensional LC and key operation modes are described:

• Heart-cutting 2D-LC: targeted transfer of defined regions from the first to the second dimension.
• Multiple heart-cutting: sequential isolation of several fractions for detailed 2D separation.
• High-resolution sampling: fine-slice heart cuts across a 1D peak to resolve coeluting species.
• Comprehensive 2D-LC: uninterrupted transfer of the complete effluent with ultra-fast cycling for full sample coverage.

Method setup is managed by a single software interface supporting gradient design for both dimensions, time- or peak-based triggering and contour plot visualization for data analysis.

Instrumentation used

• Agilent 1290 Infinity II Binary Pumps for high-pressure gradients in both dimensions.
• Bio-inert quaternary pump and autosampler with PEEK, titanium and ceramic flow paths rated to 600 bar.
• Metal-free valve modules and capillaries ensuring no nonspecific binding or corrosion.
• Optional detectors for UV/Vis, fluorescence and high-resolution MS coupling (e.g. Agilent 6530 Accurate-Mass Q-TOF).

Key results and discussion

Performance highlights from four representative case studies:

1. HILIC×RPLC-MS of mAb tryptic digests delivered higher peak capacity and clear differentiation of stressed versus native antibody samples.
2. Automated Protein A×WCX with multiple heart cuts achieved precise titer quantification and charge variant profiling without manual intervention (RSD RT < 0.3%, RSD area < 1.5%).
3. SEC×WCX combination enabled simultaneous aggregation and charge variant analysis with high reproducibility (RT RSD < 0.1%, area RSD < 1%).
4. IEX×RP-MS provided on-line desalting and intact mass qualification of rituximab variants, identifying C-terminal lysine forms with a 128 Da mass shift.
5. Comprehensive HILIC/WAX glycan mapping of fetuin and erythropoietin grouped glycans by charge in a fully automated 110 min run.
6. Time-based multiple heart-cutting of EPO glycans reduced total analysis time by over 70% compared to offline workflows.

Benefits and practical applications

• Significant increase in peak capacity and resolution for complex biopharma samples.
• Fully automated 2D workflows cut hands-on time and boost laboratory throughput.
• Flexible operation modes allow tailored assays for peptides, proteins, aggregates and glycans.
• Bio-inert, metal-free flow path preserves sensitive biomolecules across wide pH and buffer conditions.
• Seamless integration with mass spectrometry enables multi-attribute characterization in a single sequence.

Future trends and potential applications

Advances in high-speed pumps, detector sensitivity and data analytics will drive real-time monitoring of critical quality attributes during bioprocessing. Integration of 2D-LC with high-resolution MS and machine-learning-based data interpretation will further expand applications to viral vector characterization, nanoparticle formulations and complex glycoengineering studies.

Conclusion

The Agilent 1290 Infinity II 2D-LC Solution provides a versatile, high-resolution platform for automated biopharmaceutical analysis. Its bio-inert design, flexible operation modes and comprehensive software support enable robust peptide mapping, charge variant profiling, aggregation assessment and glycan mapping with minimal manual intervention.

References

• Schneider S 2D-LC/MS Characterization of Charge Variants Using Ion Exchange and Reversed-Phase Chromatography Agilent Technologies Application Note 5991-6673EN 2016
• Schneider S Online 2D-LC Characterization of Monoclonal Antibodies Using Protein A and Weak Cation Exchange Chromatography Application Note 5991-6848EN 2016
• Schneider S Online 2D-LC Characterization of Monoclonal Antibodies with Size Exclusion and Weak Cation Exchange Chromatography Application Note 5991-6906EN 2016
• Vanhoenacker G et al Analysis of Monoclonal Antibody Digests Part 2 HILIC×RPLC-MS Application Note 5991-4530EN 2014
• Schneider S Naegele E Krieger S Online 2D-LC Analysis of Complex N-Glycans in Biopharmaceuticals Application Note 5991-5349EN 2015