Characterization of Biotherapeutics: ACQUITY UPLC H-Class Bio with 2D Part 3 of 3: On-line Enrichment of Low Abundance Species

Importance of the Topic

Characterization of biotherapeutic proteins requires high resolution separation of charge variants and post-translational modifications, some of which occur at low abundance. Traditional ion exchange methods employ non-volatile buffers that hinder direct coupling to mass spectrometry and often require manual fraction collection for downstream analysis. On-line enrichment using a two-dimensional UPLC system addresses these challenges by automating fractionation, desalting, and concentration of target species for improved throughput and data quality.

Objectives and Study Overview

This study evaluates the ACQUITY UPLC H-Class Bio System with 2D Technology for on-line enrichment of low abundance charge variants in a monoclonal antibody model (infliximab). The objectives were to demonstrate automated heart-cut fractionation from ion exchange, trapping and desalting on a second reversed phase column, direct quantitation of enriched fractions, and preparation for peptide mapping.

Methodology and Instrumentation

A two-column setup was used: first dimension strong cation exchange separation with a salt gradient, followed by heart-cut transfer to a second reversed phase trap column for desalting and concentration. Valve switching sequences isolated flow paths to remove unbound salts under aqueous conditions, then eluted enriched fractions using an organic gradient. Sample preparation included deglycosylation with PNGaseF, C-terminal clipping with carboxypeptidase B, and trypsin digestion of enriched fractions.

Instrumentation Used

• ACQUITY UPLC H-Class Bio System with 2D Technology
• Quaternary Solvent Manager and Binary Solvent Manager
• ACQUITY UPLC Column Manager
• ACQUITY UPLC TUV and PDA detectors
• Protein-Pak Hi Res SP SCX column, 7 μm, 4.6×100 mm
• Protein BEH C4 trap column, 1.7 μm, 2.1×50 mm
• Peptide BEH C18 analytical column, 1.7 μm, 2.1×100 mm
• Xevo G2-S QTof mass spectrometer
• MassLynx Software v4.1 and UNIFI Scientific Information System v1.7

Key Results and Discussion

Heart-cuts of an acidic variant eluting at ~6.6 minutes were collected in sequential runs. Accumulation of up to five heart-cuts produced a proportional increase in recovered protein mass, demonstrating reproducible enrichment. Enriched fractions underwent peptide mapping, revealing deamidation of asparagine in peptide T7. MS E fragmentation localized the 1 Da mass shift to the third residue, confirming isoaspartic and aspartic acid byproducts in a 3 to 1 ratio.

Benefits and Practical Applications

This automated 2D workflow minimizes manual handling, reduces sample loss, and allows direct quantitation of enriched fractions by UV peak area. It accelerates characterization of biotherapeutics, supports identification of low abundance variants, and streamlines sample preparation for peptide mapping and MS analysis.

Future Trends and Opportunities

• Integration with higher throughput fraction collectors and multiplexed multidimensional separations
• Application to diverse biotherapeutic formats such as fusion proteins and antibody–drug conjugates
• Enhanced column chemistries to improve trapping capacity and selectivity
• Expanded use of advanced MS fragmentation techniques for comprehensive PTM profiling

Conclusion

The ACQUITY UPLC H-Class Bio System with 2D Technology provides an efficient and reproducible solution for on-line enrichment of low abundance species in complex biotherapeutic samples. Automated heart-cut fractionation, desalting, and concentration streamline workflows, improve sample recovery, and facilitate detailed peptide mapping without compromising productivity.

References

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