Isothermal chemical denaturation as a complementary tool to overcome limitations of thermal differential scanning fluorimetry in predicting physical stability of protein formulations
European journal of pharmaceutics and biopharmaceutics
Various stability indicating techniques find application in the early stage development of novel therapeutic protein candidates. Some of these techniques are used to select formulation conditions that provide high protein physical stability. Such an approach is highly dependent on the reliability of the stability indicating technique used. In this work, we present a formulation case study where we evaluate the ability of differential scanning fluorimetry (DSF) and isothermal chemical
... chemical denaturation (ICD) to predict the physical stability of a model monoclonal antibody during accelerated stability studies. First, we show that thermal denaturation technique like DSF can provide misleading physical stability ranking due to buffer specific pH shifts during heating. Next, we demonstrate how isothermal chemical denaturation can be used to tackle the above-mentioned challenge. Subsequently, we show that the concentration dependence of the Gibbs energy of unfolding determined by ICD provides better predictions for the protein physical stability in comparison to the often-used Tm (melting temperature of the protein determined with DSF) and Cm (concentration of denaturant needed to unfold 50 % of the protein determined with ICD). Finally, we give a suggestion for a rational approach which includes a combination of DSF and ICD to obtain accurate and reliable protein physical stability ranking in different formulations. Abbreviations µDSC -differential scanning microcalorimetry Cm -concentration (in M) of chemical denaturant needed to unfold 50 % of the protein ("melting" concentration of denaturant) dG -Gibbs free energy of unfolding dHionisation -enthalpy of ionisation dpH/dT -temperature dependence of pH in pH units per 1 °C dpKa/dT -temperature dependence of pKa in pH units per 1 °C DSF-differential scanning fluorimetry HMW -high molecular weight species HP-SEC -size exclusion chromatography HPW -highly purified water ICD -isothermal chemical denaturation ICH -international conference on harmonization LMW -low molecular weight species MWCO -molecular weight cut off pKa -acid dissociation constant Tm -protein melting temperature https://doi.