Tissue-type plasminogen activator (tPA) comprises six protein domains that exhibit a complex and pH-dependent thermal unfolding profile, due to partially independent domain unfolding.  Here we have used tPA as a model for evaluating the relationships between various thermal unfolding and aggregation parameters in multidomain proteins.  We show that changes in the thermal unfolding profile of tPA were parametrized by the overall thermal midpoint transition temperature, Tm, and the Van't Hoff entropy for unfolding, Δ Svh, which is a measure of unfolding cooperativity.  We used a Design of Experiments (DoE) approach to evaluate how formulation excipients impact and control the thermal unfolding profile for tPA and found that the relative stability of each of the tPA domains was dependent on the formulation. 
THERM