Fluorescence vs. UV Detection: Comparing Two CE-SDS Systems Using NISTmAb

Significance of the Topic

Protein capillary electrophoresis is essential for assessing antibody quality and purity in biopharmaceutical research and production. Fluorescence and UV detection are two major approaches, each offering unique benefits and challenges. Comparing them enhances method selection and data reliability for critical quality analytics.

Objectives and Study Overview

This study compared fluorescence detection using the Agilent ProteoAnalyzer system with UV absorbance detection on System B. By analyzing the NISTmAb monoclonal antibody reference standard, key measurements such as monomeric purity, glycan occupancy, thioether content, and fragment resolution were evaluated against each other and published reference values.

Methodology and Sample Preparation

NISTmAb samples were prepared under reduced and nonreduced conditions following respective kit manuals. Fluorescence labeling involved covalent dye conjugation and incubation at 70 °C for 10 minutes. UV analysis required no labeling but included internal markers and standard sample cleanup. Separation times were 30 minutes for the ProteoAnalyzer and 55 to 65 minutes for System B.

Instrumentation

• Agilent ProteoAnalyzer system with LED-induced fluorescence detection and Protein Broad Range P240 kit
• System B capillary electrophoresis instrument with UV absorbance detection
• Lower and upper molecular weight markers, standard capillaries, and automated analysis software

Key Results and Discussion

• Baseline Noise: Fluorescence detection showed a flat, low-noise baseline, enabling clear peak identification. UV detection exhibited baseline drift and required lamp warm-up for stability.
• Monomeric Purity: 98.18% by fluorescence vs 98.32% by UV, both within 0.3% of the 98.47% NIST value (%CV ≤ 0.19%).
• Glycan Occupancy: 99.30% by fluorescence vs 99.26% by UV, closely matching the 99.39% reference (%CV ≤ 0.02%).
• Percent Thioether: 0.40% by fluorescence vs 0.41% by UV, in agreement with the 0.30% datasheet and showing acceptable precision.
• NGHC/HC Resolution: Both systems achieved a resolution factor of 1.60, exceeding the minimum requirement and supporting accurate fragment separation.

Benefits and Practical Applications

• Fluorescence detection provides enhanced sensitivity and simplicity in peak analysis for complex protein samples.
• UV detection enables label-free measurement with minimal sample preparation.
• Both methods support routine quality control of monoclonal antibodies and compliance with regulatory standards.

Future Trends and Potential Uses

• Adoption of fully automated fluorescence workflows for increased throughput and reproducibility.
• Development of dual detection methods combining fluorescence and absorbance for comprehensive impurity profiling.
• Expansion of kit chemistries to cover wider protein size ranges and diverse sample matrices.

Conclusion

Both fluorescence detection on the Agilent ProteoAnalyzer and UV detection on System B deliver comparable accuracy and precision for NISTmAb analysis. The stable, low-noise baseline of fluorescence offers clear advantages for detailed protein quality assessment, while UV detection remains a robust, label-free alternative.

References

1. Agilent Protein Broad Range P240 Kit quick guide publication D0031125 2023
2. Agilent ProteoAnalyzer system brochure publication 5994-6716EN 2023
3. NIST Reference Material 8671 NISTmAb datasheet US Department of Commerce
4. Agilent ProteoAnalyzer technical overview publication 5994-6960EN 2024
5. Turner et al Development of Orthogonal NISTmAb Size Heterogeneity Control Methods Anal Bioanal Chem 2018 410 2095–2110