Rethinking freezing: the misconception of nucleation temperature and ice crystal formation in freeze-drying

The nucleation temperature is commonly believed to be a key factor influencing the pore size of lyophilised products. This assumption aligns with classical nucleation theory, which states that the nucleation rate, defined as the number of stable nuclei formed per unit time in a given volume, increases with the degree of supercooling, thereby producing more and smaller ice crystals in the frozen solution. During primary drying, these ice crystals sublimate, leaving behind pores that collectively form the microstructure of the lyophilisate. However, this interpretation does not fully consider a critical aspect of freezing: the exothermic nature of ice formation. This thermal event influences the resulting ice crystal structure, similar to how controlled temperature treatments, such as annealing, are purposefully used to modify ice crystal growth. To assess the impact of the temperature increase on ice crystal size distribution, a freeze-drying microscope was used in this study to compare rapidly frozen samples with those thermally treated under conditions present in a conventional freeze-dryer. The results indicate that the final ice crystal structure is not solely determined by the nucleation event, but is primarily governed by the duration of the temperature increase. However, a correlation was observed between nucleation temperature and the duration of the temperature rise, suggesting that nucleation temperature indirectly influences the freezing process. These findings challenge conventional assumptions regarding the role of nucleation temperature in freeze-drying and underscore the need for a revised perspective on ice crystal formation during freezing.