Effects of precipitation process on the biophysical properties of highly concentrated proteins

Purpose: One of the primary challenges in developing highly concentrated protein formulations is ensuring adequate stability of the protein product. Among the many available methods, cold solvent precipitation is useful, and it has been adopted for protein purification. This study aimed to characterize protein stability under various precipitation conditions and to determine the most stable precipitation conditions. Methods: Lysozyme was used as a model protein to investigate the effects of various solvents and temperature conditions on the physicochemical properties. Size exclusion chromatography, dynamic light scattering, circular dichroism, and thermodynamic analysis are the primary tools for the protein characterization. Results: Changes in the rehydrated particle size, secondary structure, thermodynamic stability, and in vitro activity of lysozyme were observed under various conditions. By analyzing the effect of temperature on protein characteristics, it has been discovered that protein precipitation at a temperature of 4 °C or below is optimal. During the precipitation at – 20 °C, the secondary structures of proteins are adversely affected by the formation of ice crystals. None of the solvents used as precipitants in this study affected the activity of the protein. However, changes in the secondary structure were minimal when ethanol and octanol were used. Conclusion: In this study, the feasibility of various precipitation conditions was evaluated for stable reversible precipitation formation. Although further studies are needed, precipitation of proteins using various organic solvents could be used in the development of formulations that purify and concentrate proteins.