Freeze-drying is the preferred method to manufacture proteins in their solid state thus the understanding of the relationship between cycle parameters and cake properties remains of great interest. The present study aims to investigate the influence of the freezing conditions in the material properties at different layers throughout the dried structure, in the presence and absence of a protein. Placebo and protein formulations were dried applying different cooling rates: slow, fast and fast cooling with annealing. Non-uniform visual cake appearance, different pore sizes and endothermic events for release of structural water were observed throughout the cake at different freezing rates indicating heterogeneous properties of the dried material likely due to heating gradients during freezing. However, annealing increased the crystallinity and eliminated material inhomogeneities across the cake. The crystalline phase was mainly comprised of δ and hemihydrate mannitol (MHH) distributed at different ratios and influenced by the presence of the protein. The undesired formation of MHH is associated to currently used freezing temperatures or amorphous to crystalline material ratios. Thus, the correlation between the freezing step parameters and resulting material structure is a step forward to provide a better understanding of the freeze-dried cake formation and product quality improvement.