Introducing automated online multicolumn two- dimensional liquid chromatography screening as a rapid and efficient tool for method development of multiple pipeline modalities

Significance of the Topic

Pharmaceutical development has shifted from predominantly small molecules to increasing amounts of complex biologics, including antibodies, engineered proteins, oligonucleotides and vaccines. Traditional one-dimensional liquid chromatography workflows struggle to resolve the structural diversity and stereoisomers present in these modalities. Automated online multicolumn two-dimensional liquid chromatography (2D-LC) screening delivers the enhanced peak capacity and selectivity required to accelerate method development for modern drug pipelines.

Objectives and Overview of the Study

This work presents an integrated platform combining online multicolumn heart-cutting 2D-LC with diode-array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS). The goals are to demonstrate rapid, automated screening of multiple column and mobile phase combinations, to separate challenging isomeric and stereoisomeric mixtures, and to confirm analyte purity in pharmaceutical pipelines.

Methodology and Instrumentation

The methodology consists of an automated 2D-LC system configured with:

• First dimension pumps, autosampler, column and DAD detector
• Multicolumn selection valves for both dimensions
• Online heart-cutting or high-resolution sampling streams
• Second dimension pump, column and DAD/ESI-MS detectors

This setup supports rapid switching among multiple reversed-phase, ion-exchange and chiral columns, along with diverse mobile phases. ESI-MS detection confirms mass identity without compromising first-dimension selectivity through MS-incompatible buffers.

Main Results and Discussion

Key demonstrations include:

• Separation of warfarin and its four hydroxylated isomers, resolving positional and chiral variants by combining achiral first-dimension retention with chiral second-dimension heart-cuts
• Discovery and structural confirmation of a major de-methylated impurity undetected by one-dimensional screening
• Automated assessment of over a hundred column/mobile phase permutations overnight, reducing manual labor and solvent consumption
• Application to protein analysis via SEC–RPLC, transferring size-exclusion fractions into a reversed-phase dimension to characterize monomer and aggregate populations

Chromatographic performance and reproducibility remained excellent across all valve and dimension switches.

Benefits and Practical Applications of the Method

This automated multicolumn online 2D-LC approach offers:

• High throughput method development through one-click screening of column/eluent combinations
• Enhanced selectivity to resolve stereoisomers, positional isomers and protein proteoforms
• Reduced project timelines by enabling overnight evaluations
• Compatibility with MS detection even when first-dimension eluents are nonvolatile

These advantages support QA/QC laboratories, analytical development groups and complex drug discovery workflows.

Future Trends and Potential Applications

Emerging directions include integration of artificial intelligence for automated method optimization, expansion to three or more dimensions for ultra-complex mixtures, coupling with high-resolution MS/MS for structural elucidation, and application to new modalities such as lipid nanoparticles and cell-derived vesicles. Implementation in regulated environments will drive broader adoption in GMP analytical pipelines.

Conclusion

The automated online multicolumn 2D-LC-DAD-ESI-MS platform described here addresses the growing analytical challenges of modern pharmaceutical development. It delivers rapid, efficient and reproducible separations across small and large molecule modalities with minimal user intervention, accelerating method development and impurity characterization.

References

• Wang H, Dunn Z, Singh A, Barrientos R, Lhotka HR, Ahmad IA, Regalado EL. Automated online multicolumn two-dimensional liquid chromatography screening for rapid method development of multiple pipeline modalities. Journal of Chromatography A. 2020;1622:460895.