Freeze-Drying of Proteins in Glass Solids Formed by Basic Amino Acids and Dicarboxylic Acids

Abstract

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 ...

Experimental evidence for excess entropy discontinuities in glass-forming solutions

“…T g itself depends on the experimental procedure, 8,53 which can lead to strong deviations in T g between different studies. In our experiments, values of 214.3 K and 218.8 K were obtained for two different concentrations, exhibiting close agreement with the values given by Kadoya et al 72 and Izutsu et al 73 The T m -and T hom -curves in Fig. 1 (dashed and dashdotted lines) are fit functions of the form of a fourth degree polynomial, omitting the third degree.…”

“…9 and 68). T m and T hom from Murray, 9 T m and T g from Maltini et al, 68 T g from Lu and Zografi, 69 ★ T g from Kadoya et al 72 and Izutsu et al 73 If not explicitly indicated in the plot, the errorbars are within the symbol size. TABLE II.…”

“…Further cooling leads to vitrification of the remaining solution at T g (see Refs. 8,72,and 73). T g itself depends on the experimental procedure, 8,53 which can lead to strong deviations in T g between different studies.…”

Component Crystallization and Physical Collapse during Freeze-Drying of <small>L</small>-Arginine–Citric Acid Mixtures

“…It was expected that the high transition temperatures (T g ′) of the L-arginine mixture frozen solutions would allow efficient primary drying at higher product temperatures. 5) However, the different molecular interactions contributing to glass formation of the disaccharide and the amino acid-carboxylic acid mixture systems require the assessment of the relationship between the thermal transition and collapse phenomena for formulation and process development. Crystallization of L-arginine in frozen aqueous solutions was also studied.…”

Stabilization of Protein Structure in Freeze-Dried Amorphous Organic Acid Buffer Salts

“…32,33) Our previous study also showed contribution of rigid molecular interactions to form glass-state solids by co-lyophilization of citric acid an L-arginine. 34) Many protein solutions have their T g Ј at temperatures (approx. Ϫ10°C) higher than those of smaller molecules.…”