TY - JOUR
T1 - Temperature stability of proteins
T2 - Analysis of irreversible denaturation using isothermal calorimetry
AU - Schön, Arne
AU - Clarkson, Benjamin R.
AU - Jaime, Maria
AU - Freire, Ernesto
N1 - Publisher Copyright:
© 2017 Wiley Periodicals, Inc.
PY - 2017/11
Y1 - 2017/11
N2 - 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.
AB - 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.
KW - chemical denaturation
KW - differential scanning calorimetry
KW - irreversible denaturation
KW - isothermal calorimetry
KW - protein denaturation and aggregation
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U2 - 10.1002/prot.25354
DO - 10.1002/prot.25354
M3 - Article
C2 - 28722205
AN - SCOPUS:85031119820
SN - 0887-3585
VL - 85
SP - 2009
EP - 2016
JO - Proteins: Structure, Function and Bioinformatics
JF - Proteins: Structure, Function and Bioinformatics
IS - 11
ER -