Peptide and antibody therapeutics are increasingly central to modern drug development, yet their formulation remains challenged by intrinsic instabilities and aggregation tendencies. In this work, we study the thermal stability of an IgG4 monoclonal antibody (mAb) in the presence of l-arginine and NaCl by combining experimental analysis with a Kirkwood-Buff theoretical framework. The antibody exhibits multistate unfolding transitions, allowing us to disentangle cosolute effects on both conformational and colloidal stability. Our results reveal a dual and concentration-dependent role of cosolutes. Accordingly, l-arginine and NaCl lower unfolding temperatures, thereby destabilizing native domains while simultaneously increasing aggregation temperatures, consistent with aggregation suppression. Contour plot analyses across protein concentrations demonstrate that aggregation inhibition is largely driven by preferential binding at the single-chain level, whereas unfolding destabilization is relatively insensitive to the protein concentration. These findings highlight the synergistic yet ambiguous influence of cosolutes or excipients such that they act as effective aggregation suppressors but promote unfolding of individual domains and vice versa. From a pharmaceutical perspective, this duality complicates rational formulation design. While cosolutes such as l-arginine enhance solubility and colloidal stability, their destabilizing impact on conformational integrity poses risks for therapeutic functionality. Our study underscores the need for careful optimization of cosolute concentrations in antibody formulations, balancing aggregation prevention with preservation of native structure. These insights provide a mechanistic basis for improved formulation strategies for the development of stable biopharmaceuticals.
Yang et al. (Fri,) studied this question.