Influence of physicochemical conditions on liquid-liquid phase separation and stability of immunoglobulin Y for storage and application

Liquid-liquid phase separation (LLPS) is a biological process and can lead to the formation of irreversible aggregates of functional proteins upon storage and administration, making it essential to predict and mitigate this phenomenon. Immunoglobulin Y (IgY), a unique class of antibody derived from egg yolk has broad applications in disease diagnosis, prophylaxis, and treatment. In this study, we observed the formation of droplet-shaped condensates of IgY under crowding conditions with polyethylene glycol 8000 (PEG 8000). To assess the relative contribution of different IgY domains to LLPS, we prepared the fragment antigen binding (Fab), fragment crystallizable (Fc) 3-4, and Escherichia coli-expressed IgY-Fc 2-4 domain. After PEG 8000 addition, the Fab fragments more propensity to aggregate, while Fc 3-4 and E. coli-expressed Fc underwent LLPS. Furthermore, we found that LLPS of IgY is influenced by electrostatic interactions. Recognizing the negative effects of LLPS on antibody efficacy, our study showed that the addition of arginine and lysine at low concentrations could prevent PEG-induced LLPS, enhancing IgY stability. These findings provide valuable insights into the optimization of IgY antibody applications and storage conditions, advancing our understanding of antibody stability in solution and facilitating the development of strategies to protect antibodies from aggregation.