Applications of isothermal titration calorimetry and differential scanning calorimetry in biopharmaceutical formulation development

One key factor in stabilization of protein drugs in liquid formulations is the choice of appropriate excipients at optimum concentrations that provide extended shelf life while also ensuring highest safety to the patient. Although general principles of stabilization have emerged from the literature over the past decade, the mechanisms by which excipients can improve the stability of a protein drug during storage are still not completely understood. Knowledge of mechanisms of protein-excipient interactions is critical as it would enable a rational approach towards optimizing protein formulations. Calorimetric investigations to explore protein-excipient interactions are increasingly being applied towards the design and optimization of biopharmaceutical formulations. Thermodynamic parameters of interactions such as excipients-protein binding and protein unfolding in the presence of excipients, assessed by using techniques such as isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC), reveal important mechanistic information needed to develop optimum formulations. Here we present two examples of how ITC and DSC can aid in protein formulation development. The first case study used ITC to reveal the interactions between polysorbate-80, a surfactant commonly used to stabilize proteins against surface adsorption and aggregation, with Protein X (ProX). The ITC data fit, using a single site model, resulted in the binding affinity constant (KA) = 1430 ± 260 M -1 , the enthalpy of binding (DH) = -6.3 ± 1.1 kcal/mol, and the number of binding sites (n) per ProX molecule = 2.6 ± 0.3. Another dataset, generated using DSC and ITC, revealed that, in the presence of the antimicrobial agent, phenol, the structural integrity of ProX was best maintained at a pH of 5.7. Thus, in these two case studies, calorimetry proved to be an elegant methodology to provide critical information useful in developing the best formulation for ProX.