Influence of Freezing Protocols and the Role of Excipients on Stability and Enzymatic Activity

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. This study focused on analyzing the effect of different cooling rates on ice crystal formation and the properties of the final product, as well as evaluating the impact of excipients such as sucrose, hydroxypropylβ-cyclodextrin (HPβ-CD), and Tween 80 in protecting the enzyme after exposure it to an accelerated stability test. The enzyme formulations under investigation included the enzyme in its native form, the enzyme with 5% w/v sucrose and 5% w/v HPβ-CD and the enzyme with 5% w/v sucrose and 0.01% w/v Tween 80. To assess the impact on enzyme stability, different freezing protocols were tested, including slow freezing at 0.1 °C/min and 0.34 °C/min, rapid freezing via liquid nitrogen immersion, and an annealing treatment applied to samples frozen at 0.1 °C/min and 0.34 °C/min. After each lyophilization cycle, the samples underwent an accelerated stability test in an oven at 50 °C for 5 and 10 days to evaluate the stability and, thus, the enzymatic activity. In parallel, reference samples were stored in a freezer at -20 °C ÷ ± 5°C for direct comparison. The characterization of the samples included the determination of residual moisture content using the Karl Fischer titration and the analysis of enzymatic activity with particular attention to temperature exposure. The study was conducted by first evaluating the impact of excipients on enzymatic activity while keeping the freezing protocol constant and subsequently assessing the influence of different freezing protocols on the same enzyme formulation. This approach enabled a detailed comparison of various experimental conditions and helped to identify the most effective strategies to stabilize peptiligase, making it more suitable for pharmaceutical and biotechnological applications. In particular, the formulation containing sucrose and Tween 80, although it tended to liquefy the final product, proved to be a good formulation for stabilizing the enzyme only when applied to a freezing protocol using liquid nitrogen. Meanwhile, the SH formulation, although less robust, was more versatile than the previous one as it tended to stabilize the enzyme regardless of the freezing protocol applied.