Online 2D-LC Analysis of Complex N-Glycans in Biopharmaceuticals Using the Agilent 1290 Infinity 2D-LC Solution

Importance of the Topic

Detailed characterization of N glycosylation on erythropoietin is essential in biopharmaceutical development because glycan heterogeneity influences drug safety efficacy and regulatory compliance. Complex sialylated structures create microheterogeneity that must be resolved for accurate isoform profiling.

Objectives and Overview

This study evaluates online two dimensional liquid chromatography concepts to achieve high resolution analysis of complex N linked glycans in therapeutic EPO. Comprehensive HILIC to WAX and multiple heart cutting WAX to HILIC workflows are compared using an Agilent 1290 Infinity 2D LC solution with fluorescence detection and QTOF mass spectrometry.

Methodology and Instrumentation

Enzymatic release of N glycans followed by 2 AB labelling enabled sensitive fluorescence detection. Two workflows were implemented:

• Comprehensive 2D LC combining HILIC in the first dimension and weak anion exchange WAX in the second dimension using a 2 position 4 port duo valve with 40 µL loops.
• Multiple heart cutting 2D LC reversing the order WAX first then HILIC second using dual 6 position 14 port valves with twelve 40 µL loops for selective peak transfers and longer second dimension gradients.

Instrumentation included Agilent 1260 Infinity Bio inert quaternary pump 1290 Infinity binary pump autosampler column compartment and valve drives coupled to a 1260 Infinity fluorescence detector and a 6530 Accurate mass Q TOF system.

Key Results and Discussion

Comprehensive HILIC WAX 2D LC achieved high peak capacity and orthogonal separation grouping glycans by charge state reflecting number of sialic acids. Peaks unresolved in one dimension were separated in the second dimension enabling clear charge profiling. Multiple heart cutting WAX HILIC allowed selection of individual glycan fractions with extended HILIC gradients revealing sub peaks below the first dimension peak shape and resolving up to eight components under a single peak region.

Benefits and Practical Applications

• Automated online workflows reduce overall analysis time by about 70 percent compared to offline fractionation.
• Detailed isoform charge profiling supports regulatory requirements and quality control of therapeutic EPO.
• Flexible method allows targeted analysis of selected glycan species with improved resolution.

Future Trends and Potential Applications

Advances in high throughput online 2D LC coupled with high resolution MS promise deeper characterization of complex glycoproteins. Integration with data analytics and machine learning could automate peak assignment and quantitation. Future developments may extend to other biotherapeutics requiring detailed glycosylation profiling and to multiplexed workflows for broader glycan libraries.

Conclusion

Online two dimensional LC solutions combining HILIC and WAX separations provide high resolution and efficient analysis of complex N glycans in erythropoietin. Comprehensive and multiple heart cutting workflows on the Agilent 1290 Infinity platform enable detailed charge profiling and peak capacity improvements while reducing analysis time. These methods support robust quality control and regulatory compliance in biopharmaceutical development.

References

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