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NIST Pathlength and Monoclonal Antibody Standards for the Quality Control of Absorbance Measurements of Protein Biopharmaceuticals



Kenneth D. Cole, Paul C. DeRose, Hua-Jun He, Erica V. Stein, Brian E. Lang, John E. Schiel, Aaron A. Urbas, Evelyn Solis, Steven J. Choquette


A rapid, and reliable method to determine protein concentration is by absorbance measurements in the ultraviolet (UV) spectrum. NIST has developed Standard Reference Material (SRM) 2082 as a pathlength standard for absorbance measurements in the UV for use with the new generation of microvolume spectrophotometers and short pathlength cuvettes. Short pathlengths are used with high concentration targets to ensure that absorbance values are within the optimal range. The short pathlength instruments and cuvettes also reduce the volumes required to conserve valuable samples. In this study, we have shown that SRM 2082 and RM 8671 (reference material, a monoclonal antibody) can be used to ensure the reliability and quality control of absorbance measurements of proteins at high concentrations, such as those used for biopharmaceutical applications. We compared the results obtained with high quality dual beam spectrophotometers and short-pathlength cuvettes to the results obtained from a microvolume spectrophotometer and a microvolume plate reader. The use of SRM 2082 can be used to increase the confidence in the measurements using the short pathlength cuvettes by calculating accurate pathlength values. RM 8671 can then be used as a system suitability control to ensure the accuracy and reproducibility of protein concentration measurements by providing an industrially relevant reference material, a well characterized monoclonal antibody.
Bioprocess Engineering


absorbance, standard, monoclonal antibody, pathlength, protein, measurement, biopharmseutical, reference material


Cole, K. , DeRose, P. , He, H. , Stein, E. , Lang, B. , Schiel, J. , Urbas, A. , Solis, E. and Choquette, S. (2018), NIST Pathlength and Monoclonal Antibody Standards for the Quality Control of Absorbance Measurements of Protein Biopharmaceuticals, Bioprocess Engineering (Accessed October 6, 2022)
Created May 8, 2018, Updated May 4, 2021