TY - JOUR
T1 - Novel non-linear curve fitting to resolve protein unfolding transitions in intrinsic fluorescence differential scanning fluorimetry
AU - Augustijn, Dillen
AU - Mahapatra, Sujata
AU - Streicher, Werner
AU - Svilenov, Hristo
AU - Kulakova, Alina
AU - Pohl, Christin
AU - Rinnan, Åsmund
PY - 2019
Y1 - 2019
N2 - In biotherapeutic protein research, an estimation of the studied protein's thermal stability is one of the important steps that determine developability as a function of solvent conditions. Differential Scanning Fluorimetry (DSF) can be applied to measure thermal stability. Label-free DSF measures amino acid fluorescence as a function of temperature, where conformational changes induce observable peak deformation, yielding apparent melting temperatures. The estimation of the stability parameters can be hindered in the case of multidomain, multimeric or aggregating proteins when multiple transitions partially coincide. These overlapping protein unfolding transitions are hard to evaluate by the conventional methodology, as peak maxima are shifted by convolution. We show how non-linear curve fitting of intrinsic fluorescence DSF can deconvolute highly overlapping transitions in formulation screening in a semi-automated process. The proposed methodology relies on synchronous, constrained fits of the fluorescence intensity, ratio and their derivatives, by combining linear baselines with generalized logistic transition functions. The proposed algorithm is applied to data from three proteins; a single transition, a double separated transition and a double overlapping transition. Extracted thermal stability parameters; apparent melting temperatures Tm,1, Tm,2 and melting onset temperature Tonset are obtained and compared with reference software analysis. The fits show R2 = 0.94 for single and R2 = 0.88 for separated transitions. Obtaining values and trends for Tonset in a well-described and automated way, will aid protein scientist to better evaluate the thermal stability of proteins.
AB - In biotherapeutic protein research, an estimation of the studied protein's thermal stability is one of the important steps that determine developability as a function of solvent conditions. Differential Scanning Fluorimetry (DSF) can be applied to measure thermal stability. Label-free DSF measures amino acid fluorescence as a function of temperature, where conformational changes induce observable peak deformation, yielding apparent melting temperatures. The estimation of the stability parameters can be hindered in the case of multidomain, multimeric or aggregating proteins when multiple transitions partially coincide. These overlapping protein unfolding transitions are hard to evaluate by the conventional methodology, as peak maxima are shifted by convolution. We show how non-linear curve fitting of intrinsic fluorescence DSF can deconvolute highly overlapping transitions in formulation screening in a semi-automated process. The proposed methodology relies on synchronous, constrained fits of the fluorescence intensity, ratio and their derivatives, by combining linear baselines with generalized logistic transition functions. The proposed algorithm is applied to data from three proteins; a single transition, a double separated transition and a double overlapping transition. Extracted thermal stability parameters; apparent melting temperatures Tm,1, Tm,2 and melting onset temperature Tonset are obtained and compared with reference software analysis. The fits show R2 = 0.94 for single and R2 = 0.88 for separated transitions. Obtaining values and trends for Tonset in a well-described and automated way, will aid protein scientist to better evaluate the thermal stability of proteins.
KW - Biotherapeutics
KW - Differential scanning fluorimetry
KW - Intrinsic fluorescence DSF
KW - Multivariate data analysis
KW - Non-linear curve fitting
KW - Protein screening
KW - Thermal stability screening
U2 - 10.1016/j.ejpb.2019.06.001
DO - 10.1016/j.ejpb.2019.06.001
M3 - Journal article
C2 - 31175923
AN - SCOPUS:85069878158
SN - 0939-6411
VL - 142
SP - 506
EP - 517
JO - European Journal of Pharmaceutics and Biopharmaceutics
JF - European Journal of Pharmaceutics and Biopharmaceutics
ER -