Sampreeti Jena1, Jacqueline Horn2, Raj Suryanarayanan3, Wolfgang Friess2, Alptekin Aksan4,5. 1. Biostabilization Laboratory, Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota, 55455, USA. 2. Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universität München, 81377, Munich, Germany. 3. Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota, 55455, USA. 4. Biostabilization Laboratory, Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota, 55455, USA. aaksan@umn.edu. 5. The BioTechnology Institute, University of Minnesota, Minneapolis, Minnesota, 55455, USA. aaksan@umn.edu.
Abstract
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.
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.