Abstract: Lyophilization is widely used in the pharmaceutical and biotechnological industries for the long-term preservation of sensitive biomolecules, such as enzymes. This process allows the removal of water through sublimation, preserving the structure and functionality of biomolecules without causing thermal damage. However, the freezing phase is a critical step, as it determines the formation and distribution of ice crystals, directly influencing the stability and activity of the enzyme after lyophilization. In this context, this thesis project, conducted at Fresenius Kabi iPSUM, a company specialized in the production and development of active pharmaceutical ingredients, aims to analyze how different freezing protocols and the addition of specific excipients influence the stability and activity of an enzyme, i.e., peptiligase.