Quantification of Structural Integrity and Stability Using Nanograms of Protein by Flow-Induced Dispersion Analysis |
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Authors: | Morten E. Pedersen,Jesper Ø stergaard,Henrik Jensen |
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Affiliation: | 1.Fida Biosystems ApS, Generatorvej 6, 2860 Soeborg, Denmark;2.Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark; |
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Abstract: | In the development of therapeutic proteins, analytical assessment of structural stability and integrity constitutes an important activity, as protein stability and integrity influence drug efficacy, and ultimately patient safety. Existing analytical methodologies solely rely on relative changes in optical properties such as fluorescence or scattering upon thermal or chemical perturbation. Here, we present an absolute analytical method for assessing protein stability, structure, and unfolding utilizing Taylor dispersion analysis (TDA) and LED-UV fluorescence detection. The developed TDA method measures the change in size (hydrodynamic radius) and intrinsic fluorescence of a protein during in-line denaturation with guanidinium hydrochloride (GuHCl). The conformational stability of the therapeutic antibody adalimumab and human serum albumin were characterized as a function of pH. The simple workflow and low sample consumption (40 ng protein per data point) of the methodology make it ideal for assessing protein characteristics related to stability in early drug development or when having a scarce amount of sample available. |
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Keywords: | FIDA protein stability protein size hydrodynamic radius Taylor dispersion analysis (TDA) protein folding automation |
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