Basic process of biological freeze-drying

Annealing is simply holding the product at a temperature above the final freezing temperature for a defined period to crystallize the potentially crystalline components (usually, crystalline bulking agent) in the formulation during the freezing stage. An annealing step is frequently necessary to allow efficient crystallization of the crystalline bulking agent, such as mannitol or glycine. Failure to crystallize the bulking agent has the potential of depressing the Tg' and compromising storage stability by crystallizing from the solid during storage. If the bulking agent crystallizes during primary drying, vial breakage may result, which is common if a high fill depth of concentrated mannitol is used. Vial breakage can be prevented by crystallization of mannitol during freezing using slow freezing or by avoiding a temperature lower than about −25°C until the mannitol has completely crystallized. Completion of crystallization may be facilitated by annealing .The annealing temperature should be between the Tg' of amorphous phase and Teu of bulking agent to give a high crystallization rate and complete crystallization. Sufficient annealing time is needed for completion of crystallization. The optimum time depends on the ratio and properties of the bulking agent used. A high mass ratio of bulking agent to other solutes (>80% of total solute, recommended) crystallizes much faster than a lower ratio (< 50% of total solute, not recommended). A low annealing temperature may tend to produce high crystallinity because supersaturation is higher at low temperature, but the crystallization rate may be too low because of high viscosity. The optimum annealing conditions are a compromise between crystallinity and crystallization rate .For mannitol or glycine, a temperature of −20 or −25°C and an annealing time of 2h or longer are suggested if the fill depth is 1 cm or more. Annealing conditions can be studied using either frozen solution X-ray diffraction or DSC procedures to evaluate the development of crystallinity. Annealing often has effects beyond crystallization of solutes. Annealing above the glass transition temperature of Tg' causes growth of ice crystals, which decreases the product resistance to flow of water vapor and results in shorter primary drying time. Also, the product specific surface area is reduced, which decreases the water desorption rate in secondary drying and may lead to increased residual moisture content in the final product or demand longer secondary drying.

Sample structure without annealing
Sample structure with annealing

Frozen sample without annealing
Frozen sample with annealing
Collapse after annealing under FDM