Proteins are essential for life. They are, among other, responsible for structure and functioning of living cells, they are elements of growth and repair, and they help us fight infections. We find them in our food, (biological) drugs, cosmetics, etc.
In solution, proteins are prone to associate. This can lead to protein crystallization, to the formation of amorphous aggregates, or to the liquid-liquid phase separation where the two liquid phases differ in the concentration of the protein. Instability of protein solutions can be both desired (e.g. protein isolation, crystallization for structure determination) and undesired (e.g. causing diseases such as cataract of the eye lens or Alzheimer‘s disease; short shelf-life of biological drugs).
The Crystalline instrument offers the possibility to quickly obtain data on the stability of protein formulations by making use of the real time digital camera. This information is vital in many industrial areas.
Lysozyme is a widely studied protein. Its aqueous solutions exhibit a complex phase behaviour dependent on the solution's composition (i.e. protein concentration, type of buffer, additives) and external conditions (temperature, pressure). In this study, Crystalline was used to monitor the phase changes upon cooling and heating aqueous-phosphate buffer lysozyme solutions in presence of different additives, such as NaCl, NaBr, NaNO3, maltose, trehalose and PEG. The cloud and clear temperature points were succesfully determined with the Crystalline instrument. Lower values of Tcloud and Tclear indicate better stability of the protein formulations. Two types of phase transitions were observed, depending on the type of additives present in the solution:
(1) Formation of crystals at Tcloud, and oiling out at Tclear.
(2) Formation of amorphous (suspension) at Tcloud, and clear solution at Tclear.
The most promissing stable behaviour was observed for the formulations using sodium chloride.
For further information please check our publications database for the application note document or the Crystalline leaflets.
Technobis Crystallization Systems is highly grateful to Tadeja Janc from University of Ljubljana for the help with this case study.