Quantification of Structural Integrity and Stability Using Nanograms of Protein by Flow-Induced Dispersion Analysis
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Quantification of Structural Integrity and Stability Using Nanograms of Protein by Flow-Induced Dispersion Analysis. / Pedersen, Morten E.; Østergaard, Jesper; Jensen, Henrik.
In: Molecules, Vol. 27, No. 8, 2506, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Quantification of Structural Integrity and Stability Using Nanograms of Protein by Flow-Induced Dispersion Analysis
AU - Pedersen, Morten E.
AU - Østergaard, Jesper
AU - Jensen, Henrik
N1 - Funding Information: Funding: Innovation Fund Denmark (grant number 9065-00009B).
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
KW - automation
KW - FIDA
KW - hydrodynamic radius
KW - protein folding
KW - protein size
KW - protein stability
KW - Taylor dispersion analysis (TDA)
U2 - 10.3390/molecules27082506
DO - 10.3390/molecules27082506
M3 - Journal article
C2 - 35458703
AN - SCOPUS:85129058893
VL - 27
JO - Molecules
JF - Molecules
SN - 1420-3049
IS - 8
M1 - 2506
ER -
ID: 306592461