Protein Aggregation in Frozen Trehalose Formulations: Effects of Composition, Cooling Rate, and Storage Temperature.

This study was designed to assess the effects of cooling rate, storage temperature, and formulation composition on trehalose phase distribution and protein stability in frozen solutions. The data demonstrate that faster cooling rates (>100°C/min) result in trehalose crystallization and protein aggregation as determined by Fourier Transform Near-Infrared (FT-NIR) spectroscopy and size-exclusion chromatography, respectively. Conversely, at slower cooling rates (≤1°C/min), trehalose remains predominantly amorphous and there is no effect on protein stability. Evaluation of storage temperatures demonstrates that aggregation increases more rapidly at -14°C compared with higher (-8°C) and lower (-20°C) storage temperatures; however, a relatively higher amount of cumulative aggregation was observed at lower (-20°C) temperature compared with higher storage temperatures (-14°C and -8°C). Further evaluation of the effects of formulation composition suggests that the phase distribution of amorphous and crystallized trehalose dihydrate in frozen solutions depends on the ratio of trehalose to mAb. The results identify an optimal range of trehalose-mAb (w/w) ratio, 0.2-2.4, capable of physically stabilizing mAb formulations during long-term frozen storage-even for fast cooled (>100°C/min) formulations.