Analysis of phase behavior and morphology during freeze-thaw applications of lysozyme

Knowledge of protein behavior/stability during freeze/thaw (FT) operations is essential for storage and production processes in the biopharmaceutical industry. FT stress involves freeze concentration, cold denaturation, and ice crystals formation which can result in protein aggregation. Therefore, it is important to understand the ongoing FT processes, and the influence of different solution parameters. In order to evaluate the ongoing processes during FT (up to -80°C), phase diagrams with lysozyme from chicken egg white and sodium chloride were generated. Thereby, three different buffer systems with varying buffer substances and ionic strengths at pH 3 and pH 5 were investigated. As indicators for the ongoing FT processes, the phase behavior, crystal morphology and solubility were used. An increased number of cycles led, for example, to the formation of micro crystals, sea urchin crystals - indicating LLPS and/or high supersaturation - and precipitate. Furthermore, the buffer substances had a more distinct influence on the phase behavior and morphology compared to the ionic strength differences. The solubility line itself was only shifted when distinct changes in the phase behavior could be observed. In summary, a tool was developed for using the phase behavior and especially the crystal morphology as indicator for underlying processes during FT operations.