The freezing step in lyophilization: physico-chemical fundamentals, freezing methods and consequences on process performance and quality attributes of biopharmaceuticals.

Lyophilization is a common, but cost-intensive, drying process to achieve protein formulations with long-term stability. In the past, typical process optimization has focused on the drying steps and the freezing step was rather ignored. However, the freezing step is an equally important step in lyophilization, as it impacts both process performance and product quality. While simple in concept, the freezing step is presumably the most complex step in lyophilization. Therefore, in order to get a more comprehensive understanding of the processes that occur during freezing, the physico-chemical fundamentals of freezing are first summarized. The available techniques that can be used to manipulate or directly control the freezing process in lyophilization are also reviewed. In addition, the consequences of the freezing step on quality attributes, such as sample morphology, physical state of the product, residual moisture content, reconstitution time, and performance of the primary and secondary drying phase, are discussed. A special focus is given to the impact of the freezing process on protein stability. This review aims to provide the reader with an awareness of not only the importance but also the complexity of the freezing step in lyophilization and its impact on quality attributes of biopharmaceuticals and process performance. With a deeper understanding of freezing and the possibility to directly control or at least manipulate the freezing behavior, more efficient lyophilization cycles can be developed, and the quality and stability of lyophilized biopharmaceuticals can be improved.