Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability

Purpose: The physical state of excipients in freeze-dried formulations directly affects the stability of the active pharmaceutical ingredient (API). Crystallization of trehalose and mannitol in frozen solutions has been shown to be a function of composition. However, to date a detailed study of the effect of concentrations of the API and other excipients on the crystallinity of mannitol and trehalose in frozen solutions has not been reported. Methods: The crystallinity of mannitol and trehalose in frozen solutions was characterized by Differential Scanning Calorimetry, X-ray diffractometry, and FTIR spectroscopy. The secondary structure of BSA was probed by FTIR, and Circular Dichroism spectroscopy in frozen and thawed solutions, respectively. Results: Trehalose crystallization was accompanied by unfolding of BSA. BSA delayed and reduced the extent of mannitol and trehalose crystallization. Similar effects were observed upon adding D₂O (≥5% w/w) and low concentrations of polysorbate 20 (≤0.2% w/w) with retention of BSA in its native conformation. At high BSA to trehalose mass ratio, the protein could stabilize itself in the frozen state, but unfolded upon thawing. Conclusions: The API and other excipients, in a concentration-dependent manner, influenced the physical state of the freeze concentrate as well as the stability of the API.