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A Comparison of Protein Quantitation Assays for Biopharmaceutical Applications

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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-iT™) 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.

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Notes

  1. Certain commercial materials, instruments, and equipment are identified in this manuscript in order to specify the experimental procedure as completely as possible. In no case does such identification imply recommendation or endorsement by the National Physical Laboratory, nor does it imply that the material, instrument or equipment identified is necessarily the best available for the purpose.

Abbreviations

AAA:

Amino acid analysis

ANOVA:

Analysis of variance

BCA:

Bicinchoninic acid

BSA:

Bovine serum albumin

CBQCA™:

3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde

CD:

Circular dichroism

CV:

Coefficient of variance

Da:

Dalton

FTIR :

Fourier transformed infrared (spectroscopy)

ICH:

International Conference on Harmonisation

IEX:

Ion exchange

LOD:

Limit of detection

Mw:

Relative molecular weight

PITC:

Phenylisothiocyanate

PEG:

Polyethylene glycol

PEG-5000 SE:

O-Methyl-O’-succinylpolyethylene glycol 5000 N-succinimidyl ester

σ:

Standard deviation

S/N:

Signal/noise ratio

UV:

Ultraviolet

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Acknowledgements

The work was funded by the UK’s Department of Trade and Industry’s National Measurement System through the Measurements for Biotechnology (MfB) programme. We wish to thank George Tranter and Alison Rodger for comments on the manuscript.

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Authors and Affiliations

  1. National Physical Laboratory, Hampton Road, Teddington, Middlesex, TW11 0LW, UK

    J. E. Noble, A. E. Knight & M. J. A. Bailey

  2. M-Scan Ltd, 3 Millars Business Centre, Fishponds Close, Wokingham, Berkshire, RG41 2TZ, UK

    A. J. Reason & A. Di Matola

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  1. J. E. Noble
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  2. A. E. Knight
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  3. A. J. Reason
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  4. A. Di Matola
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  5. M. J. A. Bailey
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Correspondence to J. E. Noble.

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Noble, J.E., Knight, A.E., Reason, A.J. et al. A Comparison of Protein Quantitation Assays for Biopharmaceutical Applications. Mol Biotechnol 37, 99–111 (2007). https://doi.org/10.1007/s12033-007-0038-9

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