Feasibility of a cryopreservation of cultured human corneal endothelial cells.

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/mm2) gave a similar cell density for cryopreserved HCECs to that of non-preserved control HCECs after 28 days of cultivation (2099 cells/mm2 and 2111 cells/mm2, 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.

Freeze-drying of proteins with glass-forming oligosaccharide-derived sugar alcohols.

Physical properties and protein-stabilizing effects of sugar alcohols in frozen aqueous solutions and freeze-dried solids were studied. Various frozen sugar alcohol solutions showed a glass transition of the maximally freeze-concentrated phase at temperatures (T(g)'s) that depended largely on the solute molecular weights. Some oligosaccharide-derived sugar alcohols (e.g., maltitol, lactitol, maltotriitol) formed glass-state amorphous cake-structure freeze-dried solids. Microscopic observation of frozen maltitol and lactitol solutions under vacuum (FDM) indicated onset of physical collapse at temperatures (T(c)) several degrees higher than their T(g)'s. Freeze-drying of pentitols (e.g., xylitol) and hexitols (e.g., sorbitol, mannitol) resulted in collapsed or crystallized solids. The glass-forming sugar alcohols prevented activity loss of a model protein (LDH: lactate dehydrogenase) during freeze-drying and subsequent storage at 50 degrees C. They also protected bovine serum albumin (BSA) from lyophilization-induced secondary structure perturbation. The glass-forming sugar alcohols showed lower susceptibility to Maillard reaction with co-lyophilized L-lysine compared to reducing and non-reducing disaccharides during storage at elevated temperature. Application of the oligosaccharide-derived sugar alcohols as alternative stabilizers in lyophilized protein formulations was discussed.

Stabilization of protein structure in freeze-dried amorphous organic acid buffer salts.

The purpose of this study was to elucidate the physical properties and protein-stabilizing effects of some pH-adjusting excipients (carboxylic acids and their sodium salts) in frozen solutions and in freeze-dried solids. Thermal and powder X-ray diffraction (XRD) analysis indicated a high propensity of sodium citrates to form glass-state amorphous solids upon freeze-drying. Some salts (e.g., sodium succinate) crystallized in the single-solute frozen solutions. FT-IR analysis of bovine serum albumin (BSA) and bovine immunoglobulin G (IgG) in the aqueous solutions and the freeze-dried solids showed that some glass-forming salts (e.g., monosodium citrate) protected the secondary structure from lyophilization-induced perturbation. Freeze-drying of BSA at different concentrations indicated retention of the secondary structure at similar monosodium citrate/protein concentration ratios, suggesting stabilization through direct interaction that substitute water molecules inevitable for the conformation integrity. The carboxylic acid salts should provide rigid hydrogen bonds and electrostatic interactions that raise the glass transition temperature of the amorphous solids and stabilize protein structure. The relevance of the structural stabilization to the protein formulation design was discussed.

Freeze-drying of proteins in glass solids formed by basic amino acids and dicarboxylic acids.

The purpose of this study was to produce and characterize glass-state amorphous solids containing amino acids and organic acids that protect co-lyophilized proteins. Thermal analysis of frozen solutions containing a basic amino acid (e.g., L-arginine, L-lysine, L-histidine) and a hydroxy di- or tricarboxylic acid (e.g., citric acid, L-tartaric acid, DL-malic acid) showed glass transition of maximally freeze-concentrated solute at temperatures (T'g) significantly higher than those of the individual solute solutions. Mixing of the amino acid with some dicarboxylic acids (e.g., oxalic acid) also suggested an upward shift of the transition temperature. Contrarily, combinations of the amino acid with monocarboxylic acids (e.g., acetic acid) had T'gs between those of the individual solute solutions. Co-lyophilization of the basic amino acids and citric acid or L-tartaric acid resulted in amorphous solids that have glass transition temperatures (Tg) higher than the individual components. Mid- and near-infrared analysis indicated altered environment around the functional groups of the consisting molecules. Some of the glass-state excipient combinations protected an enzyme (lactate dehydrogenase, LDH) from inactivation during freeze-drying. The glass-state excipient combinations formed by hydrogen-bonding and electrostatic interaction network would be potent alternative to stabilize therapeutic proteins in freeze-dried formulations.

Glass-state amorphous salt solids formed by freeze-drying of amines and hydroxy carboxylic acids: effect of hydrogen-bonding and electrostatic interactions.

We studied effect of molecular interactions on the physical properties of binary freeze-dried solids and frozen aqueous solutions using model chemicals containing various functional groups (amino, carboxyl, hydroxyl). Thermal analysis of frozen solutions containing alkyl diamines and hydroxy di- or tricarboxylic acids showed thermal transitions (T(g)': glass transition of maximally freeze-concentrated phase) at temperatures higher than those of the individual solutes. A binary frozen solution containing 80 mM 1,3-diamino-2-hydroxypropane (single-solute T(g)'<-60 degrees C) and 120 mM citric acid (single-solute T(g)': -55.0 degrees C) made the transition at -30.8 degrees C. The molecular weight of the solutes had smaller effects on the transition temperatures of the frozen mixture component solutions. Lyophilization of some high T(g)' mixture frozen solutions (e.g., 1,3-diamino-2-hydroxypropane and citric acid) resulted in cake-structure amorphous solids with glass transition temperatures (T(g)) higher than those of the individual components. Networking of intense hydrogen-bondings and electrostatic interactions between the heterogeneous molecules through the multiple functional groups was suggested to reduce the component mobility in the amorphous freeze-concentrated phase and the freeze-dried solids. Controlling the interactions should be a key to optimizing the physical properties of multi-component amorphous freeze-dried pharmaceutical formulations.