Determination of Protein Extinction Coefficients and Concentration by UV‑Vis

Importance of the Topic

Accurate determination of protein extinction coefficients and concentrations is essential in biochemistry and biopharmaceutical development. UV-Vis spectroscopy at 280 nm provides a rapid, non-destructive approach to quantify proteins by exploiting the absorbance of aromatic amino acids. Reliable measurements support critical processes from drug discovery to manufacturing and quality control of mAbs and vaccines.

Objectives and Study Overview

This study evaluates the performance of the Agilent Cary 3500 Multicell UV-Vis spectrophotometer combined with Agilent Cary Workstation software for:

• Automated determination of protein extinction coefficients via the Beer–Lambert law.
• Sequence-based estimation of molar extinction coefficients.
• Comparison of extinction coefficients for an innovator mAb (Ristova) and two biosimilars (Reditux, Truxima).
• Generation of a calibration curve to estimate mAb concentrations in process samples.

Methodology and Used Instrumentation

Protein samples (lysozyme, β-casein, ovalbumin, Ristova, biosimilars) were prepared in 6 M guanidine hydrochloride. Measurements were performed at 280 nm with baseline correction at 320 nm.

Used Instrumentation

• Agilent Cary 3500 Multicell UV-Vis Spectrophotometer with permanently focused beam.
• Multicell Peltier UV-Vis detector module, 2.00 nm spectral bandwidth.
• Ultra-microvolume rectangular cuvettes (10 mm path, 70 µL).
• Agilent Cary Workstation Software for automated equation calculation and calibration curves.

Main Results and Discussion

Extinction coefficients determined by the instrument matched sequence-based values and literature reports with deviations below 5%. The software’s built-in equation editor calculated ε values for multiple proteins simultaneously. Comparison of Ristova, Reditux, and Truxima at 1 and 0.5 mg/mL showed identical ε values within experimental error, confirming similar aromatic amino acid compositions. A calibration curve for Ristova over 0.1–2 mg/mL yielded R²=0.9918, demonstrating linear response and suitability for bioprocess monitoring.

Benefits and Practical Applications

• High throughput measurement of up to eight samples per run without alignment.
• Automated data analysis reduces operator time and error.
• Accurate protein concentration estimation supports bioprocess development and QC.
• Sequence-based and experimental ε determination ensures analytical robustness.

Future Trends and Opportunities

Integration of UV-Vis spectrophotometry with advanced data analytics, cloud-based workflows, and real-time process monitoring will further enhance lab efficiency. Miniaturization of optical systems and expanded wavelength coverage can enable multi-parametric assays for emerging biotherapeutics and complex mixtures.

Conclusion

The Agilent Cary 3500 Multicell UV-Vis spectrophotometer paired with Cary Workstation software offers a powerful solution for rapid, accurate protein quantification. Automated extinction coefficient calculations, combined with sequence-based validation and robust calibration curves, streamline workflows in biopharmaceutical research and production. Comparison of innovator and biosimilar mAbs confirmed the method’s reliability for assessing molecular similarity.

Reference

1. Gill SC, von Hippel PH. Calculation of Protein Extinction Coefficients from Amino Acid Sequence Data. Anal Biochem. 1989;182(2):319–326.
2. Lee KH et al. Analytical Similarity Assessment of Rituximab Biosimilar CT-P10 to Reference Medicinal Product. MAbs. 2018;10(3):380–396.
3. Petrat-Melin B et al. In Vitro Digestion of Purified Β-Casein Variants: Effects on Antioxidant and ACE Inhibitory Capacity. J Dairy Sci. 2015;98(1):15–26.