43Freeze-drying is a popular method of ensuring the stability of proteins that are not stable enough in aqueous solutions during the period required for storage and distribution. 1,2) Various freeze-dried protein formulations contain excipients (e.g., sugars, polymers, and amino acids) that protect proteins from physical and chemical changes. Disaccharides (e.g., sucrose, trehalose) are the most popular among them because they stabilize proteins both thermodynamically and kinetically in aqueous solutions and freeze-dried solids. [3][4][5] The development of freeze-dried protein formulations containing amino acids is often more challenging than the development of formulations with saccharides because of the varied physical and chemical properties (e.g., crystallinity, glass transition temperature) of the freeze-dried amino acids, as well as their tendency to form complexes with other ingredients. 6) Many amino acids are considered to protect proteins basically in similar mechanisms with disaccharides. They thermodynamically stabilize protein conformation in aqueous solutions and probably in frozen solutions by being preferentially excluded from the immediate surface of proteins. 7) Glass-state amorphous solids formed by freeze-drying of the disaccharides or some amino acids protect proteins from structural changes thermodynamically by substituting surrounding water molecules. 8) They also reduce chemical degradation of freeze-dried proteins kinetically by reducing the molecular mobility. 2,8) In addition, some amino acids (e.g., L-arginine) also prevent protein aggregation in aqueous solutions prior to the drying process and after reconstitution. 9) Choosing appropriate counterions that form glass-state solid should be one of the key factors in designing amino acid-based amorphous freeze-dried formulations. 10,11) For example, glass transition temperatures (T g ) of freeze-dried Lhistidine salts depend largely on the counterions. 12) Colyophilization of L-arginine and multivalent inorganic acids (e.g., H 3 PO 4 , H 2 SO 4 ) results in glass-state amorphous solids that protect proteins during the process and storage (e.g., tissue plasminogen activator formulation, PDR 2003). 13) Some organic acid and inorganic cation combinations (e.g., sodium citrates) also form high glass transition temperature amorphous solids. 14) Various functional groups (e.g., amino, carboxyl, hydroxyl) in the constituting molecules contributes significantly to form the glass-state amorphous salt solids. 15) Producing glass-state amorphous solids by freeze-drying of amino acid and organic acid combinations, and their application in pharmaceutical formulations are interesting topics to explore. 15) The purpose of this study was to produce stable amorphous solids that protect proteins by freeze-drying combinations of amino acids and organic acids. The physical properties of frozen solutions and freeze-dried solids containing the popular excipients and model chemicals were studied. The effect of the excipient combinations on the freeze-drying ...

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821Glass-state amorphous solids are applied to pharmaceutical formulations as a way to improve dissolution of hydrophobic active ingredients (APIs) or to ensure stability of embedded biomacromolecules (e.g., recombinant proteins) and drug delivery system (DDS) carriers (e.g., liposome). 1-3)Freeze-drying is often a preferable method over other procedures (e.g., quench-cooling of heat-melt solids) for the large-scale production of glass-state solid formulations containing thermally unstable ingredients. Dispersion of drug molecules into nonionic excipient matrices (e.g., trehalose, polyvinylpyrrolidone (PVP)) is a popular way to make the non-crystalline formulations of many APIs that have intrinsic propensity for crystallization or low glass transition temperatures (T g ).1) Insufficient miscibility with certain drug molecules and poor storage stability, however, often hinders the development of solid dispersion formulations using the nonionic matrices.The application of salts or binary complexes is another approach to obtain stable amorphous solids. 4) For example, freeze-drying of sodium indomethacin results in an amorphous solid that has a glass transition temperature (120°C) significantly higher than that of the free acid molecules (45°C). 4,5) Recent studies indicated that the glass-state solids composed of excipient salts or salt-forming excipient combinations are promising as dispersion matrices. 6,7) Some pHadjusting buffer salts (e.g., monosodium citrate) form high T g amorphous solids applicable to protein formulations. 7) Colyophilization of some basic amino acids (e.g., L-arginine, Llysine, L-histidine) with multivalent inorganic acids (e.g., phosphoric acid) also results in the formation of protein-stabilizing glass-state solids.6) High transition temperatures of the mixture freeze-dried solids suggest the contribution of strong intermolecular or inter-ion interactions to reducing the heterogeneous component mobility.In contrast to the extensive studies on the physical properties and local structure of amorphous glass-or rubber-state solids composed of nonionic small molecules (e.g., sucrose, sorbitol) or polymers (e.g., PVP), 3) mechanisms that determine character of organic salts and/or heterogeneous components have not been well elucidated.1,3) Recent intensive studies on ionic liquids (RTMS: room temperature molten salts) provided valuable information regarding the component ion structures, participating interactions, and the physical properties of the microscopically unordered non-crystalline salt systems.8) Some earlier studies indicated feasibility to control the physical property of the amorphous salt solids by optimizing the component structure (e.g., ion radius in indomethacin alkali metal salts) 4) and their compositions that determine the intermolecular and/or inter-ion interactions.The purpose of this study was to elucidate the contribution of functional groups and the size of consisting molecules to the physical properties of multi-component frozen aqueous solutions and their freeze-dri...

The development of protein pharmaceuticals requires rational formulation design to ensure appropriate storage stability, because the degradation of such pharmaceuticals through various chemical and physical pathways not only reduces their therapeutic effects but also increases the risk of product immunogenicity. [1][2][3][4][5] Freeze-drying is a popular method of conferring long-term stability of therapeutic proteins that is not achievable in aqueous solutions. Removal of the surrounding water molecules during the freeze-drying process, however, often perturbs the protein structure, leading to irreversible aggregation in the reconstituted solutions. The structurally altered protein molecules are also prone to chemical degradation during storage.1) Maintaining the protein conformation by process and ingredient (e.g., stabilizer, pH buffer, isotonic agents) optimization thus improves both the physical and chemical stability of protein formulations.Choosing the solution pH and buffer system appropriate to a particular protein is a simple but significant element in the formulation design because the chemical and physical integrity of proteins in the aqueous solutions and freeze-dried solids depend largely on the pH. 6) Some buffer components also favorably or adversely affect the protein stability through direct interactions and/or through changing the local environments in the dried state. For example, freezing of certain buffer systems (e.g., sodium phosphate) often induces crystallization of a component salt and resulting shift of the local pH surrounding the proteins.7-11) Freeze-drying from some buffer systems (e.g., L-histidine, citrate, or Tris) often leads to higher activity retention of proteins (e.g., coagulation factor VIII, recombinant human interleukin-1 receptor antagonist) relative to those from other buffers.12-15) Conformation of the proteins lyophilized in these buffer systems is of particular interest.Reported properties of some carboxylic acid salts, including stabilization of native protein conformation in aqueous solutions (e.g., antithrombin III) 16,17) and their propensity to form glass-state amorphous solids upon lyophilization, 18) suggest their ability to protect protein conformation against dehydration stress through mechanisms similar to disaccharides. Non-reducing disaccharides (e.g., sucrose, trehalose) are popular stabilizers in solution and freeze-dried protein formulations. Various saccharides and polyols thermodynamically favor native protein structures over denatured states in aqueous solutions by a "preferential exclusion" mechanism. 19) Sucrose and trehalose protect proteins by substituting surrounding water molecules through hydrogen bonds during the freeze-drying process. 4,[20][21][22] Limited molecular mobility in glass-state lyophilized disaccharide solids also protects embedded proteins from chemical degradation (e.g., deamidation) during storage. 23)The present study assesses the physical properties and protein-stabilizing effects of carboxylic acid buffer systems (e.g.,...

Transparency of the cornea is essential for vision and is maintained by the corneal endothelium. Consequently, corneal endothelial decompensation arising from irreversible damage to the corneal endothelium causes severe vision impairment. Until recently, transplantation of donor corneas was the only therapeutic choice for treatment of endothelial decompensation. In 2013, we initiated clinical research into cell-based therapy involving injection of a suspension of cultured human corneal endothelial cells (HCECs), in combination with Rho kinase inhibitor, into the anterior chamber. The aim of the present study was to establish a protocol for cryopreservation of HCECs to allow large-scale commercial manufacturing of these cells. This study focused on the effects of various cryopreservation reagents on HCEC viability. Screening of several commercially available cryopreservation reagents identified Bambanker hRM as an effective agent that maintained a cell viability of 89.4% after 14 days of cryopreservation, equivalent to the cell viability of 89.2% for non-cryopreserved control cells. The use of Bambanker hRM and HCECs at a similar grade to that used clinically for cell based therapy (passage 3–5 and a cell density higher than 2000 cells/mm 2 ) gave a similar cell density for cryopreserved HCECs to that of non-preserved control HCECs after 28 days of cultivation (2099 cells/mm 2 and 2111 cells/mm 2 , respectively). HCECs preserved using Bambanker hRM grew in a similar fashion to non-preserved control HCECs and formed a monolayer sheet-like structure. Cryopreservation of HCECs has multiple advantages including the ability to accumulate stocks of master cells, to transport HCEC stocks, and to manufacture HCECs on demand for use in cell-based treatment of endothelial decompensation.