Sumie Yoshioka

Decreased Protein-Stabilizing Effects of Cryoprotectants Due to Crystallization

The stabilizing effects of various additives against inactivation of an enzyme (β-galactosidase from Aspergillus oryzae) during freeze-drying were studied, with a focus on their crystallinity. The crystalline morphology of mannitol and inositol in freeze-dried cakes depended on the solute concentrations before freezing and the freeze-drying method used. The additives in their amorphous state showed concentration-dependent stabilization of the enzyme, whereas additive crystallization during freeze-drying decreased their effects. Heat treatment before freeze-drying also caused crystallization and diminished the stabilizing effects. Noncovalent soluble aggregates were observed in the inactivated enzyme solution. These results show the importance of maintaining the amorphous state of additives used as stabilizing agents during freeze-drying.

Preparation of poly(l-lactide) microspheres of different crystalline morphology and effect of crystalline morphology on drug release rate

Abstract The physicochemical properties of progesterone-loaded poly(l-lactide) microspheres prepared by the solvent evaporation method were studied, focusing on the crystallinity of the polymer matrices. The solvent evaporation process was found to govern the physical characteristics of microspheres. Solvent removal at atmospheric pressure yielded microspheres of crystalline polymer matrices, while faster solvent removal under a reduced pressure gave microspheres of amorphous polymer matrices. The crystallinity of the polymer matrices was closely correlated with the morphology and physical properties of microspheres, and affected the drug release rate. The microspheres of crystalline polymer matrices had rough surfaces with large surface areas, and exhibited a rapid drug release. In contrast, the microspheres of amorphous polymer matrices had smooth surfaces with smaller surface areas, and provided a slower drug release. The results of X-ray powder diffraction, differential scanning calorimetry and Fourier-transform infra-red spectroscopy suggested that progesterone formed a molecular dispersion in the amorphous polymer matrices.

Effect of temperature on mechanisms of drug release and matrix degradation of poly(d,l-lactide) microspheres

Abstract Drug release and matrix degradation of poly ( d,l -lactide) microspheres with different glass transition temperatures ( T g ) were investigated at various temperatures in order to clarify the effect of temperature on mechanisms of drug release and matrix degradation. At temperatures above T g , the average molecular weight of the polymer decreased markedly during drug release. Progesterone release was faster than microsphere weight loss, and could be fitted to the Higuchi equation. These results suggest that diffusion from the bulk of the matrix contributed to drug release at temperatures above T g . In contrast, at temperatures below the T g of the microspheres, the average molecular weight of the polymer did not change throughout the experimental period and matrix degradation was restricted to the matrix surface. Release of progesterone was due mainly to surface erosion. These results suggest that, even in the case of polylactide, drug release can be controlled only by surface erosion.

Effects of sugars and polymers on crystallization of poly(ethylene glycol) in frozen solutions: Phase separation between incompatible polymers

AbstractPurpose. This study examined the effect of third components (low-molecular-weight saccharides and polymers) on the crystallization of poly(ethylene) glycol (PEG) in frozen solutions, focusing on the relationship between their crystallization-inhibiting ability and molecular compatibility. Methods. Effects of sugars and polymers on the crystallization of PEG 3000 in frozen solution were monitored by differential scanning calorimetry (DSC). Pulsed-NMR was employed to monitor the molecular mobility of water and solutes in the frozen solutions. Miscibility between PEG and third components in aqueous solution was estimated from the lowering of cloud point of PEG 20,000. Thermal analysis of frozen solutions containing some non-crystallizing solutes was used to examine the possibility of phase separation in frozen solutions. Results. Some sugars and polymers inhibited the crystallization of PEG and formed practically stable amorphous phases among ice crystals. The mobility of solute molecules in the amorphous phase increased above the softening temperature of maximally concentrated solutions (Ts), whereas that of water molecules appeared at a lower temperature. Mono- and disaccharides that are relatively less miscible with PEG in solution inhibit PEG crystallization to a lesser degree. Two Ts regions were observed in frozen solutions containing both polyvinylpyrrolidone (PVP) and dextran, at much lower concentrations than those causing aqueous two-phase separation at ambient temperatures. Conclusions. Ice crystallization raises the concentration of solutes in the remaining solution, which can lead to phase separation in the amorphous phase. Molecular compatibility between components is an important factor determining their propensity to phase separate and crystallize.

The effect of excipients on the molecular mobility of lyophilized formulations, as measured by glass transition temperature and NMR relaxation-based critical mobility temperature.

AbstractPurpose. The dependence of the molecular mobility of lyophilized formulations on pharmaceutical polymer excipients was studied. Molecular mobility as determined by NMR relaxation-based critical temperature of molecular mobility (Tmc) and glass transition temperature (Tg) is discussed in relation to the plasticizing effect of water in formulations. Methods. The Tmc and Tg of lyophilized γ-globulin formulations containing 6 different polymer excipients such as dextran, polyvinylpyrrolidone (PVP) and methylcellulose (MC) was determined by NMR and DSC. The molecular mobility of water in the formulations was determined by proton NMR and dielectric relaxation spectrometry (DRS). Results. Tmc varied with polymer excipients. Tmc increased as the ratio of bound water to mobile water increased and as the molecular mobility of mobile water decreased. The formulation containing MC exhibited a lower Tmc than the formulation containing dextran because of the smaller ratio of bound water and the higher molecular mobility of mobile water. The Tmc of the formulation containing PVP was higher than that expected from the higher T2 values of water because of the lower molecular mobility of mobile water regardless of the higher ratio of mobile water. The Tmc of these lyophilized formulations was higher than their Tg by 23°C to 34°C, indicating that the formulations became a NMR-detected microscopically liquidized state below their Tg. Conclusions. The quantity and the molecular mobility of mobile water in lyophilized formulations can be considered to affect the Tmc of lyophilized formulations, which in turn governs their stability.

Physical stability and protein stability of freeze-dried cakes during storage at elevated temperatures

The relationship between physical stability of freeze-dried cakes and protein stability during storage was studied using β-galactosidase as a model protein and inositol as an excipient. Amorphous samples freeze-dried from solutions containing the enzyme and various concentrations of inositol in sodium phosphate buffer (50 mM, pH 7.4) were stored for 7 days over P2O5 at 40 to 70°C. Structural collapse and inositol crystallization were observed in some of the samples, depending on the formulation and storage temperature. The physical stability of freeze-dried samples was also studied by differential scanning calorimeter (DSC). Inositol showed a protein-stabilizing effect when its amorphous form was retained during storage, regardless of structural collapse. However, crystallization of inositol during storage removed its stabilizing effect. Addition of water-soluble polymers such as dextran, Ficoll and carboxymethyl cellulose sodium salt (CMC-Na) preserved activity of the enzyme by preventing inositol crystallization.

Increased Stabilizing Effects of Amphiphilic Excipients on Freeze-Drying of Lactate Dehydrogenase (LDH) by Dispersion into Sugar Matrices

AbstractPurpose. The stabilizing effect of amphiphilic excipients and sugars against protein inactivation during freeze-drying was studied in relation to their physical states in freeze-dried cakes. Methods. Physical states of amphiphilic excipients were studied by powder X-ray diffractometry and differential scanning calorimetry (DSC). Stabilizing effects of excipients were studied using lactate dehydrogenase (LDH) as a model protein. Results. Although poly(ethylene glycols) (PEGs) 1000 to 20000 crystallized when freeze-dried alone, the addition of sugars decreased their crystallinity by dispersing PEG into sugar-dominant matrices. Sugars species, molecular weight of PEGs, and buffer concentration also affected the crystallinity of PEGs. Sugars also dispersed some of other amphiphilic excipients, which tended to crystallize or become “microscopically liquid” when freeze-dried without sugar. Only the amphiphilic excipients that remained amorphous solid state protected the enzyme during freeze-drying in the absence of sugars. However, combinations of sucrose and all the amphiphilic excipients studied increased the stabilizing effects markedly. The remaining activities were greater than the sum of their individual ones. Conclusions. Various amphiphilic excipients are good stabilizers for freeze-drying of proteins when dispersed into sugar-dominant matrices.

Dependence of the Molecular Mobility and Protein Stability of Freeze-Dried γ-Globulin Formulations on the Molecular Weight of Dextran

AbstractPurpose. The effect of the molecular weight of dextran on the molecular mobility and protein stability of freeze-dried serum γ-globulin (BGG) formulations was studied. The stabilizing effect of higher molecular weight dextran is discussed in relation to the molecular mobility of the formulations. Methods. The molecular mobility of freeze-dried BGG formulations containing dextrans of various molecular weights was determined based on the free induction decay of dextran and water protons measured by proton NMR. The protein stability of the formulations was determined at temperatures ranging from 20 to 70°C by size exclusion chromatography. Results. Changes in the molecular mobility of freeze-dried formulations that occurred at temperatures below the glass transition temperature could be detected as the molecular mobility-changing temperature (Tmc), at which dextran protons started to exhibit a Lorentzian relaxation decay due to higher mobility in addition to a Gaussian relaxation decay. Tmc increased as the molecular weight of dextran increased. The proportion of dextran protons which exhibited the higher mobility relaxation process (Phm) at temperatures above Tmc decreased as the molecular weight of dextran increased. Protein stability was closely related to molecular mobility. The temperature dependence of the denaturation rate changed at around Tmc, and denaturation in the microscopically liquidized state decreased as Phm decreased with increasing molecular weight of dextran. Conclusions. The effect of the molecular weight of dextran on the protein stability of freeze-dried BGG formulations could be explained in terms of the parameters obtained by 1H-NMR such as Tmc and Phm. These parameters appear to be useful in preformulation and stability prediction of freeze-dried formulations.

The Aggregation of Bovine Serum Albumin in Solution and in the Solid State

Abstract— The aggregation of bovine serum albumin (BSA) in the solid state and in solution was studied to elucidate the effect of water mobility on the aggregation rate and mechanism. The results suggest that the freeze‐dried BSA forms covalently‐bonded aggregates via disulphide bonding during storage in the moistened solid state and in solution. The aggregation rate largely depended on the water content. The freeze‐dried BSA to which a small amount of water was added (in the moistened state) was found to be more liable to aggregation than that in solution. The aggregation rate in the moistened solid state exhibited a maximum at a very low level of moisture. In contrast, the aggregation rate in solution increased with increasing ratio of water to protein (with decreasing protein concentration). The results suggest that the increased rate is related to the increase in water mobility, as measured by the spin‐lattice relaxation time, T1, of water using 17O NMR, with increasing ratio of water to protein.

Usefulness of the Kohlrausch- Williams-Watts Stretched Exponential Function to Describe Protein Aggregation in Lyophilized Formulations and the Temperature Dependence Near the Glass Transition Temperature

AbstractPurpose. We studied the feasibility of using the Kohlrausch-Williams-Watts stretched exponential function (KWW equation) to describe protein aggregation in lyophilized formulations during storage. Parameters representing “mean aggregation time” (τa) and stretched exponential constant (βa) were calculated according to the KWW equation by assuming that the time required for protein molecules to aggregate (τ) varies because of the fact that protein aggregation occurs at a rate that depends on the degree of protein deformation resulting from stresses created during freeze-drying. The temperature dependence of the parameters near the glass transition temperature was examined to discuss the possibility of predicting protein aggregation by accelerated testing. Methods. Protein aggregation in lyophilized bovine serum γ-globulin (BGG) formulations containing dextran or methylcellulose, at temperatures ranging from 10 to 80°C, was followed by size-exclusion chromatography. Results. Non-exponential BGG aggregation in lyophilized formulations could be described by the KWW equation. The τa and βa parameters changed abruptly around the NMR relaxation-based critical mobility temperature for formulations containing dextran and methylcellulose. In the glassy state, in contrast, the τa parameter of these formulations exhibited continuous temperature dependence. The parameter τΓ, as calculated from τa and βa, reflected differences in τ values between the two excipients. Conclusions. The results indicate that the parameter βa is reflective of physical changes wihtin lyophilized formulations. Within the temperature range, during which no abrupt changes in βa were observed, knowledge regarding the τaand βa parameters allows the rate of protein aggregation to be predicted. The parameter τΓ was found to be useful in comparing the protein aggregation behavior of formulations having different τa and βa values.