Novel Non-linear Curve Fitting to Resolve Protein Unfolding Transitions in Intrinsic Fluorescence Differential Scanning Fluorimetry.

Novel Non-linear Curve Fitting to Resolve Protein Unfolding Transitions in Intrinsic Fluorescence Differential Scanning Fluorimetry.
Eur J Pharm Biopharm. 2019 Jun 05;:
Authors: Augustijn D, Mahapatra S, Streicher W, Svilenov H, Kulakova A, Pohl C, Rinnan Å
Abstract
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 ...

Source: European Journal of Pharmaceutics and Biopharmaceutics - June 4, 2019 Category: Drugs & Pharmacology Authors: Augustijn D, Mahapatra S, Streicher W, Svilenov H, Kulakova A, Pohl C, Rinnan Å Tags: Eur J Pharm Biopharm Source Type: research