Freeze-thaw of pharmaceutical solutions: counter-intuitive finding of an increase in mechanical stress between Tg” and Tg’ in frozen sucrose/water mixtures

High-resolution synchrotron X-ray powder diffraction has been utilized to detect mechanical stresses in frozen solutions, via analysis of the profiles of the diffraction peaks of ice. Increase in the width of the peaks (peaks broadening) reflects disruption of the crystal lattice, with contributions including a decrease in the crystallite size and an increase in microstrain. Frozen sucrose solutions (5 and 10 % w/v) were frozen at 100 K and then heated and annealed at -45 °C (228 K) and -15 °C (258 K), i.e. between the two apparent glass transition events in the freeze-concentrated solutions (Tg" and Tg'), and above the Tg', respectively. A decrease in microstrain and an increase in ice crystallite size were observed during annealing at -15 °C (above the Tg'), which is consistent with Ostwald ripening of ice crystals. Unexpectedly, and for the first time, an opposite trend was observed during annealing at the lower temperature of -45 °C, between Tg" and Tg'. To the best of our knowledge, this is the first report of an increased strain in the crystalline ice domains and of simultaneous size reduction during annealing of frozen aqueous solutions. Considering that the interaction with ice crystals may result in protein destabilization, the size and microstrain of ice crystallites can serve as markers of the freeze/thaw stresses on biopharmaceuticals. A practical implication is that a prolonged hold between Tg" and Tg' may increase stress imposed on protein molecules, i.e. a lower temperature is not always better for preserving biopolymers when freezing their aqueous solutions.