Novel non-linear curve fitting to resolve protein unfolding transitions in intrinsic fluorescence differential scanning fluorimetry

Research output: Contribution to journalJournal articleResearchpeer-review

Dillen Augustijn, Sujata Mahapatra, Werner Streicher, Hristo Svilenov, Alina Kulakova, Christin Pohl, Åsmund Rinnan

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.

Original languageEnglish
JournalEuropean Journal of Pharmaceutics and Biopharmaceutics
Volume142
Pages (from-to)506-517
Number of pages12
ISSN0939-6411
DOIs
Publication statusPublished - 2019

    Research areas

  • Biotherapeutics, Differential scanning fluorimetry, Intrinsic fluorescence DSF, Multivariate data analysis, Non-linear curve fitting, Protein screening, Thermal stability screening

ID: 228365797