Amorphous–Amorphous Phase Separation of Freeze-Concentrated Protein and Amino Acid Excipients for Lyophilized Formulations

Abstract

The objective of this study was to elucidate the mixing state of proteins and amino acid excipients concentrated in the amorphous non-ice region of frozen solutions. Thermal analysis of frozen aqueous solutions was performed in heating scans before and after a heat treatment. Frozen aqueous solutions containing a protein (e.g., recombinant human albumin, gelatin) or a polysaccharide (dextran) and an amino acid excipient (e.g., L-arginine, L-arginine hydrochloride, L-arginine monophosphate, sodium L-glutamate) at varied mass ratios showed single or double T g ′ (glass transition temperature of maximally freeze-concentrated solutes). Some mixture frozen solutions rich in the polymers maintained the single T g ′ of the freeze-concentrated amorphous solute-mixture phase. In contrast, amino acid-rich mixture frozen solutions revealed two T g ′s that suggested transition of concentrated non-crystalline solute-mixture phase and excipient-dominant phase. Post-freeze heat treatment induced splitting of the T g ′ in some intermediate mass ratio mixture solutions. The mixing state of proteins and amino acids varied depending on their structure, salt types, mass ratio, composition of co-solutes (e.g., NaCl) and thermal history. Information on the varied mixing states should be valuable for the rational use of amino acid excipients in lyophilized protein pharmaceuticals.Key words amorphous; freeze drying; thermal analysis; protein formulation; stabilization; mixing Freeze-drying is a popular method to formulate therapeutic proteins that are insufficiently stable in aqueous solutions during storage.1) Several freeze-dried therapeutic protein formulations contain multiple excipients, including stabilizers, bulking agents, pH buffer agents, and tonicity modifiers besides active pharmaceutical ingredient (API).2-4) Sucrose and trehalose are popular stabilizers that protect proteins from dehydration-induced irreversible structural changes during the process and from chemical degradation during storage. Certain amino acids and their salts are potent stabilizers in the freeze-drying of proteins, frozen storage of liposomes, and spray drying of vaccines.5-11) Application of the amino acid excipients that protect proteins through particular mechanisms not achievable by saccharides (e.g., aggregation-reducing effect of L-arginine (L-Arg) in aqueous solution) would increase formulation strategies in lyophilization of marginally stable proteins. 5,8,[12][13][14][15] Physical states (e.g., crystallinity, crystal polymorph) of the components are important factors that determine chemical and conformational stability of proteins during the freeze-drying process and subsequent storage. [16][17][18] The stabilizing effects of disaccharides are attributed to protection of protein conformation by the substitution of surrounding water molecules and by holding protein molecules in lower molecular mobility glassstate amorphous solids. Crystallization of some sugar alcohols (e.g., mannitol) during the freezing segment of lyophilization deprives the...

Inhomogeneous Distribution of Components in Solid Protein Pharmaceuticals: Origins, Consequences, Analysis, and Resolutions

“…44,52 Thermal history (such as annealing or the freeze-drying or spray-drying condition) affects immiscibility differently through changing component mobility. 7 Annealing in freeze-drying which raises product temperature above the Tg 0 (the glass transition temperature of the maximally freeze-concentrated solutions) to produce a good cake structure can introduce sufficient molecular mobility to solutes thereby promoting phase separation. 53,54 Elevated storage temperature and humidity can compromise the amorphous state of the formulation.…”

“…58 Frozen mixtures of protein (recombinant human albumin, gelatin) or polysaccharide (dextran) and various amino acids (L-arginine, L-arginine hydrochloride, L-arginine monophosphate, sodium L-glutamate) exhibited phase separation when NaCl or buffers were added to the formulation. 7,45 The effect is concentration-dependent and could be the result of altered hydration states in macromolecules or concealment of the electrostatic effects between them. 7,55,58 Adsorption at Various Surfaces for Surface-Active Components Limited co-solubility is not the only origin of component inhomogeneity.…”

“…7,45 The effect is concentration-dependent and could be the result of altered hydration states in macromolecules or concealment of the electrostatic effects between them. 7,55,58 Adsorption at Various Surfaces for Surface-Active Components Limited co-solubility is not the only origin of component inhomogeneity. As proteins are generally surface-active, they can be adsorbed at various interfaces formed during the drying process.…”

“…Characteristics of thermal transitions like the temperature, shape, and number of transitions are important for sample assessment and provide information on the structural arrangement of components in a solid. 7,55 The introduction of modulated DSC further improved the sensitivity to record thermal transitions by separating the heat flow that does and does not respond to a changing heating rate also referred to as reversing and non-reversing heat flow, respectively. Because most formulations after freeze-drying and spray-drying are amorphous, the Tg is primarily used to evaluate components miscibility.…”

“…[1][2][3] Typical excipients for solid protein pharmaceuticals are di-and polysaccharides, other types of polymers, and amino acids. 1,[4][5][6][7] Freeze-drying or spray-drying promotes the formation of a stabilizer matrix in the glassy state, protecting the protein. Other additives like buffers, surfactants, and antioxidants are also often combined to further enhance the stability of solid protein pharmaceuticals.…”