Isothermal chemical denaturation as a complementary tool to overcome limitations of thermal differential scanning fluorimetry in predicting physical stability of protein formulations

Abstract

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 approach is highly dependent on the reliability of the stability indicating technique used. In this work, we present a formulation case study in which we evaluate the ability of differential scanning fluorimetry (DSF) and isothermal chemical denaturation (ICD) to predict the physical stability of a model monoclonal antibody during accelerated stability studies. First, we show that a thermal denaturation technique like DSF can provide misleading physical stability rankings 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 free energy of unfolding determined by ICD provides better predictions for the protein physical stability in comparison to the often-used T (melting temperature of the protein determined with DSF) and C (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.

Considerations for the Design of Antibody-Based Therapeutics

“…Recently, the ability of thermal unfolding assays to predict physical stability has been questioned, with properties such as self-association, colloidal stability, and chemical denaturation stability being proposed as superior predictive parameters than melting temperature. 313,315 Tryptophan fluorescence is another property potentially correlated to total monomer loss (fragmentation plus aggregation) rather than aggregation alone. 313 The presence of aggregationprone regions may also be predicted using sequence-based computational modeling approaches.…”

Orthogonal Techniques to Study the Effect of pH, Sucrose, and Arginine Salts on Monoclonal Antibody Physical Stability and Aggregation During Long-Term Storage

“…Such pH dependence of thermal unfolding and aggregation is already reported for several other monoclonal antibodies. 21,[29][30][31] We then focused on the effect of several additives on the stability of PPI13 at pH 5.0 and pH 6.5 because the protein behaves differently in these conditions concerning its thermal unfolding and aggregation.…”

A New Approach to Study the Physical Stability of Monoclonal Antibody Formulations—Dilution From a Denaturant

“…Furthermore, non-isothermal techniques suffer from the fact that the properties of many excipients (e.g. the pH of amine buffers like histidine) change during heating, which can affect the obtained stability rankings based on protein melting temperatures 7 .…”

“…Isothermal chemical denaturation (ICD) is a valuable technique that avoids the above-mentioned drawbacks of DSC and DSF 5,7 . Historically, ICD was not the method of choice for protein formulation studies mostly due to the tedious sample preparation 8 .…”

“…Historically, ICD was not the method of choice for protein formulation studies mostly due to the tedious sample preparation 8 . These limitations of ICD have recently been overcome by the use of equipment that can (semi-)automatically prepare and measure protein samples containing varying concentrations of a denaturating agent 5,[7][8][9][10] . However, the accurate thermodynamic evaluation of ICD data assumes that the protein unfolding process is fully reversible, the system is in equilibrium and the denaturation graph fits a known model (e.g.…”