Enhancing Robustness for Biopharmaceutical Separations Using the Waters ACQUITY UPLC PLUS Series

Significance of the Topic

Liquid chromatography plays a central role in biopharmaceutical development and quality control. Consistent, high-resolution separations of biomolecules such as glycans, protein aggregates, and peptide digests are critical for ensuring product safety, efficacy, and regulatory compliance. Enhancing robustness and uptime of LC platforms can accelerate drug pipelines and reduce operational costs.

Objectives and Study Overview

This application note evaluates the performance equivalency and robustness improvements of Waters ACQUITY UPLC PLUS Series systems compared to legacy ACQUITY UPLC platforms. Three common biopharmaceutical workflows were tested: glycan profiling, size exclusion chromatography (SEC), and peptide mapping. Each PLUS system was directly compared to its conventional counterpart under identical conditions.

Methodology and Instrumentation

The study compared:

• ACQUITY UPLC H-Class Bio vs. H-Class PLUS Bio for glycan profiling and SEC.
• ACQUITY UPLC I-Class vs. I-Class PLUS for peptide mapping.

Chromatographic methods used included HILIC for released glycans, phosphate-buffered SEC for protein aggregates, and reversed-phase gradients for peptide digests. Key consumables and hardware:

• BEH Glycan Amide, Protein BEH SEC, and BEH C18 columns.
• RapiFluor-MS glycan standards, BEH200 protein mix, and MassPREP Enolase digest standard.
• Enhanced solvent manager and sample manager modules featuring next-generation degasser, improved mixing, and advanced needle surface treatment.

Main Results and Discussion

Glycan profiling:

• Both H-Class and H-Class PLUS systems produced identical elution profiles of RapiFluor-MS glycans.
• PLUS system reduced retention-time RSD from 0.105% to 0.082%.

Size exclusion chromatography:

• Five-component protein mixture resolved in 3 min with matching retention times.
• Retention-time RSD improved from 0.098% to 0.056% on PLUS system.

Peptide mapping:

• 75 min shallow gradient yielded equivalent separation of enolase peptides.
• I-Class PLUS reduced retention-time RSD from up to 0.044% to 0.014%.

Benefits and Practical Applications

The ACQUITY UPLC PLUS Series maintains established method performance while enhancing reproducibility and uptime. Laboratories gain confidence in data consistency across isocratic and gradient applications without method re-validation. Reduced carryover and improved thermal control benefit sensitive samples and high-throughput workflows.

Future Trends and Opportunities

Next-generation LC platforms will likely integrate further automation, real-time diagnostics, and AI-driven method optimization. Advances in sample handling, solvent delivery, and detector sensitivity may extend robust separations to emerging biotherapeutics, including complex glycoforms and large protein assemblies.

Conclusion

The ACQUITY UPLC PLUS Series delivers equivalent chromatographic performance to legacy platforms while significantly improving robustness. Demonstrated reductions in retention-time variability across glycan profiling, SEC, and peptide mapping highlight its suitability for rigorous biopharmaceutical workflows, enabling reliable data and streamlined operations.

References

1. Cosgrave EFJ, Lauber MA, Birdsall R, McCarthy SM. Combined RapiFluor-MS Labeling and UPLC H-Class Bio for N-Glycan Monitoring. Waters App Note. 2015;720005352EN.
2. Cosgrave EFJ, McCarthy SM. Automating pH Consistency in SEC Methods for Biopharma QC. Waters App Note. 2015;720005264EN.
3. Simeone J, Hong P, McConville PR. Performance of UPLC I-Class PLUS for Long Shallow Gradients. Waters App Note. 2018;720006290EN.