Protein Quantification in Formulation Buffer Using a BioResolve RP mAb Polyphenyl Column

Significance of the Topic

Protein quantification in therapeutic formulations is essential for drug product quality control and research. Reversed-phase liquid chromatography can overcome limitations of UV absorbance methods when buffers or excipients interfere with measurements.

Objectives and Study Overview

This study evaluates a BioResolve RP mAb Polyphenyl column for quantifying monoclonal antibodies and fusion proteins in histidine buffer containing recombinant human serum albumin (rHSA). Targets include rituximab, interleukin-12, erythropoietin and abatacept.

Methodology and Instrumentation

Separations were performed on a Waters ACQUITY UPLC H-Class Bio system using a BioResolve RP mAb Polyphenyl column (2.1×50 mm, 2.7 µm, 450 Å) with UV detection at 280 nm. Mobile phases were 0.1% trifluoroacetic acid in water (A) and acetonitrile (B). Gradient profiles, flow rates (0.2–1.8 mL/min) and column temperatures (60–80 °C) were optimized per analyte. Data were acquired with Empower 3 software.

Results and Discussion

Proteins were baseline or well resolved from histidine and rHSA in under 10 minutes with resolutions ≥3.5. Calibration curves and linear dynamic ranges:

• Interleukin-12: 0.01–2 µg at 0.3 mL/min (error <8%); 0.1–5 µg at 1.8 mL/min (error <10%).
• Erythropoietin: 0.5–10 µg (error <3%).
• Abatacept: 0.2–5 µg (error <3%).
• Rituximab: 0.1–5 µg at 80 °C (error <12%).

Injection-to-injection carryover was ≤0.2%, ensuring reliable high-throughput quantification.

Benefits and Practical Applications

This approach delivers rapid, high-resolution separation of therapeutic proteins in complex buffers without interference from formulation excipients. The low carryover and wide dynamic ranges support accurate quantification in QA/QC and research laboratories.

Future Trends and Applications

Emerging directions include coupling with mass spectrometry for enhanced specificity, development of affinity-based stationary phases for selective capture, and further miniaturization to increase throughput and reduce solvent consumption.

Conclusion

The BioResolve RP mAb Polyphenyl column offers a robust, efficient platform for quantifying protein drugs in histidine-based formulations containing rHSA, overcoming the limitations of direct UV absorbance methods.

References

1. Nguyen JM et al. Designing a new particle technology for reversed-phase separations of proteins. Waters Application Note 720006168EN, 2018.
2. Nguyen JM et al. A Novel Phenyl-Based RPLC Stationary Phase for High Throughput, High Resolution Characterization of Protein Therapeutics. Waters Application Note 720006169EN, 2018.
3. Zbacnik TJ et al. Role of buffers in protein formulations. Journal of Pharmaceutical Sciences. 2017;106:713–733.
4. Human Serum Albumin as a Pharmaceutical Excipient. Drug Development Web resource.
5. Hamza T et al. Interleukin 12, a key immunoregulatory cytokine in infection applications. International Journal of Molecular Sciences. 2010;11:789–806.
6. Erythropoietin. Wikipedia entry.
7. Abatacept. Wikipedia entry.
8. Rituximab. Wikipedia entry.