Stability of Protein Formulations and Their Importance
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. They are a growing area of interest within the pharmaceutical sector. In solution, proteins are prone to associate. This can lead to protein crystallization, to the formation of amorphous aggregates, or to liquid-liquid phase separation where the two liquid phases differ in the concentration of the protein. Phase transition in protein formulations may appear due to many factors: type of the protein and its concentration, pH and temperature of the solution, type and amount of additives (e.g. salts, sugars). 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.
Lysozyme is a widely studied protein. Its aqueous solutions exhibit complex phase behaviour dependent on the solution’s composition (i.e. protein concentration, type of buffer, additives) and external conditions such as temperature, pressure. In this study, the Crystalline instrument was used to monitor the phase changes upon cooling and heating aqueous-phosphate buffer lysozyme solutions in presence of different additives such as sodium chloride (NaCl), sodium bromide (NaBr), sodium nitrate (NaNO3), maltose, trehalose and PEG 10 000 (polyethylene glycol). Cloud temperature point, Tcloud, was determined upon cooling the solution. These were determined using the Crystalline instruments real time digital camera to easily and accurately detect the phase transition.
Results and Conclusions
It was found that the Tcloud and Tclear decrease when salts are added to lysozyme solutions. Lower values of Tcloud and Tclear indicate better stability of the protein formulations. NaCl showed the best results followed by NaBr and NaNO3. Similarly, the Addition of sugars (maltose and trehalose) decreases the Tcloud and Tclear stabilizing the formulation. However, addition of PEG 10 000 (polyethylene glycol) increases the Tcloud and Tclear destabilizing the formulation. These results are summeriszed in table 1.
Using the images captured on the in situ cameras, 2 types of phase transitions were observed depending on the type of additive present in the solution(see Figure 1).
(1) Formation of crystals at Tcloud, and emulsion - oiling out at Tclear.
(2) Formation of amorphous (suspension) at Tcloud, and clear solution at Tclear