Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2007 Oct;37(2):99-111.
doi: 10.1007/s12033-007-0038-9.

A comparison of protein quantitation assays for biopharmaceutical applications

Affiliations
Comparative Study

A comparison of protein quantitation assays for biopharmaceutical applications

J E Noble et al. Mol Biotechnol. 2007 Oct.

Abstract

Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications, some typical of biopharmaceuticals have been used to assess the utility of dye-based protein concentration assays. The protein concentration assays (Bicinchoninic acid (BCA), Bradford, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), DC, Fluorescamine and Quant-i) were compared to the 'gold standard' assay, quantitative amino acid analysis (AAA). The assays that displayed the lowest variability between proteins, BCA and DC, also generated improved estimates when BSA was used as a standard, when compared to AAA derived concentrations. Assays read out by absorbance tended to display enhanced robustness and repeatability, whereas the fluorescence based assays had wider quantitation ranges and lower limits of detection. Protein modification, in the form of glycosylation and PEGylation, and the addition of excipients, were found to affect the estimation of protein concentration for some of the assays when compared to the unmodified protein. We discuss the suitability and limitations of the selected assays for the estimation of protein concentration in biopharmaceutical applications.

PubMed Disclaimer

References

    1. Adv Drug Deliv Rev. 1999 Feb 1;35(2-3):289-306 - PubMed
    1. Anal Biochem. 1995 Mar 20;226(1):191-3 - PubMed
    1. Methods Enzymol. 1975;35:221-6 - PubMed
    1. Mol Biotechnol. 2003 Jan;23(1):19-28 - PubMed
    1. Bioconjug Chem. 2000 Mar-Apr;11(2):195-201 - PubMed

Publication types

LinkOut - more resources