During the downstream process and formulation of proteins, certain lag and storage phases are unavoidable. Thus, proteins are frozen in order to achieve higher long term stability. The stress put on the protein during this process can result in decreased stability and a loss of conformation. In order to increase stability during freezing and thawing, understanding the mechanisms of destabilization during that process is of paramount importance. Well-known influencing parameters on stability are cryoprotectants, buffer components and pH value. Additionally, the process of freezing and thawing itself can be optimized by using ramps with different temperature change rates and lag periods.<br/>The stability of protein solutions can be assessed using phase diagrams. The final phase behavior and the morphology, observed after 40 days at 20 °C, indicate different protein-protein and/or protein-solvent interactions, which are influenced by the previously mentioned parameters. By varying these parameters in high-throughput screenings, conclusions about their effect on interactions and protein conformation can be drawn. This method is useful for the better understanding of freezing and thawing process and how to influence it. For buffer substances, the pH and additives like salt, the influences are well understood and to some extend predictable. The influence of different freezing and thawing ramps is not understood and has to be investigated.<br/>This project aimed at analyzing the effect of different freezing and thawing ramps on the phase behavior of lysozyme from chicken egg white. Phase diagrams were prepared in a small scale using either different freezing or thawing ramps in order to observe the effects of freezing and thawing separately. It was shown that freezing and thawing influences phase behavior, solubility and morphology of lysozyme. A number of one to three cycles were performed to simulate several freezing and thawing steps. Both tested parameters could be shown to have a significant impact on the solubility and morphology.

Influence of different freeze and thaw ramps on the phase behavior of lysozyme from chicken egg white

255th National Meeting (2018)

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