The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein‐protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B22), a parameter of solution non‐ideality, which is obtained using self‐interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B22 was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B22 is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B22 decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein‐protein interactions. The B22 parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting‐in/salting‐out effect. It is also shown that B22 correlates with protein solubility as well as physical protein stability.
Lysozyme-lysozyme interactions were assessed in the native state at 25 degrees C as well in the denatured state at 80 degrees C by affinity chromatography in order to measure the osmotic second virial coefficient (B). This parameter allows us to better understand protein aggregation pathways and colloidal protein stability. Repulsive interactions (B > 0) were weakened for both protein states by increasing salt concentration and by increasing the pH value toward lysozyme pI. This decrease was more pronounced in the denatured state, most likely caused by changes in electrostatic interactions and the formation of hydrophobic clusters. The lysozyme formulations presenting the more repulsive conditions (B > 0), as derived from the osmotic second virial coefficient, showed better colloidal stability under mechanical and thermal stresses. As expected, B values are much more negative for the interactions in the denatured state compared to the data obtained for the native state, reflecting a strong tendency of denatured lysozyme to aggregate. Thus, measurement of protein interactions by affinity chromatography allows us to gain information on protein interactions in both native and denatured states as well as to predict solution conditions prone for improving protein colloidal stability.
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