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Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry
Author(s) -
Schön Arne,
Clarkson Benjamin R.,
Jaime Maria,
Freire Ernesto
Publication year - 2017
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.25354
Subject(s) - chemistry , denaturation (fissile materials) , isothermal titration calorimetry , enthalpy , isothermal process , calorimetry , protein aggregation , lysozyme , precipitation , protein stability , differential scanning calorimetry , kinetics , protein folding , thermodynamics , chromatography , biochemistry , nuclear chemistry , quantum mechanics , meteorology , physics
The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (∼100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day −1 . Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.