Design of self-interaction chromatography as an analytical tool for predicting protein phase behavior

Solution conditions under which proteins have a tendency to crystallize correspond to a slightly negative osmotic second virial coefficient (B ₂₂). A positive B₂₂ value guarantees no crystallization to occur. On the other hand, a B₂₂ value within the so called "crystallization slot" thermodynamically supports the crystallization processes but does not guarantee successful crystal growth. It is, however, a prerequisite for protein crystallization that the B₂₂ value must be in the slightly negative regime. Self-interaction chromatography (SIC) is designed in this work as an analytical tool for determining B₂₂ in a precise and reproducible way. The methodology was demonstrated in detail in terms of its theoretical basis, experimental methodology, troubleshooting and data analysis for different protein samples and solution conditions. The inherent error limit of SIC is found to be comparatively less than other B ₂₂ measurement techniques. The designed experimental approach was applied for mapping crystallization conditions of a model protein, i.e. lysozyme. Good agreement between the obtained lysozyme B₂₂ values and literature values confirms the accuracy of the approach.