Predictions of Onset of Crystallization from Experimental Relaxation Times I-Correlation of Molecular Mobility from Temperatures Above the Glass Transition to Temperatures Below the Glass Transition

Purpose Predicting onsets of crystallization at temperatures below T _g, from data above T _g, would require that the correlation between crystallization onset and mobility is same above and below T _g, and the techniques being used to measure mobility above and below T _g are measuring essentially the same kind of mobility. The aim of this work is to determine if the relaxation times obtained using different techniques (DSC, TAM) below T _g correlate with relaxation time obtained above T _g using dielectric spectroscopy.Methods Model compounds for this work were chosen based on their varied ΔH _f, ΔC _p(T _g) and H-bonding in crystalline state vs. amorphous state. Relaxation times above T _g were determined by the simultaneous fit of real and imaginary permittivity to the Cole-Davidson model. Tau and beta below T _g were determined using isothermal microcalorimetry (TAM) or MDSC. MDSC was used to calculate Kauzmann temperature and strength of the glass using established relationships.Results Indomethacin, nifedipine and flopropione showed Arrhenius temperature dependence throughout the entire temperature range and extrapolation of τ ^β measured above T _g by dielectric relaxation agreed with τ ^β measured below T _g by TAM/MDSC. Ketoconazole, however, showed the expected VTF behavior. For at least two compounds compared (indomethacin and ketoconazole), relaxation times measured by TAM and MDSC did not agree, with TAM giving significantly lower values of τ ^β , but TAM and MDSC relaxation times appeared to extrapolate to a common value at T _g.Conclusions It was found that, for all cases studied, relaxation time constants determined above and below T _g did appear to extrapolate to the same value around T _g indicating that molecular mobility measured above and below T _g using different techniques is highly correlated.     

Evaluation of Crystallization Behavior on the Surface of Nifedipine Solid Dispersion Powder Using Inverse Gas Chromatography

Purpose

To investigate crystallization behavior on the surface of amorphous solid dispersion powder using inverse gas chromatography (IGC) and to predict the physical stability at temperatures below the glass transition temperature (T _g).

Methods

Amorphous solid dispersion powder was prepared by melt-quenching of a mixture of crystalline nifedipine and polyvinylpyrrolidon (PVP) K-30. IGC was conducted by injecting undecane (probe gas) and methane (reference gas) repeatedly to the solid dispersion at temperatures below T _g. Surface crystallization was evaluated by the retention volume change of undecane based on the observation that the surface of the solid dispersion with crystallized nifedipine gives an increased retention volume.

Results

On applying the retention volume change to the Hancock-Sharp equation, surface crystallization was found to follow a two-dimensional growth of nuclei mechanism. Estimation of the crystallization rates at temperatures far below T _g using the Avrami-Erofeev equation and Arrhenius equation showed that, to maintain its quality for at least three years, the solid dispersion should be stored at ?20°C (T _g???65°C).

Conclusions

IGC can be used to evaluate crystallization behavior on the surface of a solid dispersion powder, and, unlike traditional techniques, can also predict the stability of the solid dispersion based on the surface crystallization behavior.     

Using the low-frequency Raman spectroscopy to analyze the crystallization of amorphous indomethacin

This paper gives a detailed analysis of the low-frequency Raman spectrum (LFRS) in the 5–250 cm⁻¹ region, corresponding to collective vibrations, in the crystalline forms and in the amorphous state of indomethacin (IMC). This study points out the high sensitivity of the LFRS to detect, identify and evaluate the first traces of crystallization in comparison with high-frequency regions where internal vibration bands are detected. This analysis reveals that amorphous IMC prepared by cryogrinding instantaneously partially crystallizes at room temperature in the stable γ phase, well below T_g = 43 °C. A method based on the treatment of the LFRS to determine precise and very low volume of crystallized material within amorphous matrix is described and used to analyze the crystallization kinetics of ground amorphous IMC powder. This study demonstrates that Raman spectroscopy is also a well-adapted technique to point out small amount of amorphous state in crystalline matrix. Crystallization of ground IMC powder was also analyzed by isothermal microcalorimetry experiments, which is one of the most widely used methods to analyze isothermal crystallization and to evaluate crystallinity.     

Fast Surface Crystallization of Amorphous Griseofulvin Below <Emphasis Type="Italic">T</Emphasis><Subscript>g</Subscript>

Purpose  

To study crystal growth rates of amorphous griseofulvin (GSF) below its glass transition temperature (T _g) and the effect of surface crystallization on the overall crystallization kinetics of amorphous GSF.     

Prediction of Onset of Crystallization in Amorphous Pharmaceutical Systems: Phenobarbital,Nifedipine/PVP,and Phenobarbital/PVP

The aim of this work is to determine if a stability testing protocol based on the correlations between crystallization onset and relaxation time above the glass transition temperature (T_g) can be used to predict the crystallization onsets in amorphous pharmaceutical systems well below their T_g. This procedure assumes that the coupling between crystallization onset and molecular mobility is the same above and below T_g. The stability testing protocol has been applied to phenobarbital, phenobarbital/polyvinylpyrrolidone (PVP) (95/5, w/w), and nifedipine/PVP (95/5, w/w). Crystallization onsets have been detected by polarized light microscopy examination of amorphous films; molecular mobility has been determined by dielectric relaxation spectroscopy above T_g and by both isothermal calorimetry and modulated differential scanning calorimetry below T_g. We find that small amounts of PVP significantly retard re-crystallization. This dramatic effect of PVP is not related to mobility, so this approach applies, at best, to extrapolation of high temperature data on a given formulation to low temperatures. Variation in molecular mobility at these concentrations of PVP is not the dominant factor in determining variation in propensity for re-crystallization from glassy systems; we suggest surface interactions between PVP and nuclei and/or small crystals slowing growth control variation in crystallization kinetics between formulations. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3887-3900, 2010     

Probing Beta Relaxation in Pharmaceutically Relevant Glasses by Using DSC

Purpose This study was conducted to demonstrate the use of differential scanning calorimetry (DSC) in detecting and measuring β-relaxation processes in amorphous pharmaceutical systems. Methods DSC was employed to study amorphous samples of poly(vinylpyrrolidone) (PVP), indomethacin (IM), and ursodeoxycholic acid (UDA) that were annealed at temperatures (T_a) around 0.8 of their glass transition temperatures (T_g). Dynamic mechanical analysis (DMA) was used to measure β-relaxation in PVP. Results Reheating the annealed samples gives rise to annealing peaks that occur below T_g. The peaks cannot be generated when annealing below the low temperature limit of β-relaxation. These limits are around 50°C for PVP, −20°C for IM, and 30°C for UDA. The effective activation energy (E) of the sub-T_g relaxation has been estimated for each T_a and found to increase with T_a, reflecting increasing contribution of the α-process. Estimates of E for β-relaxation have been obtained from the lowest T_a data, and are as follows: 68 (PVP), 56 (IM), 67 (UDA) kJ mol⁻¹. Conclusions DSC can be used for detecting β-relaxation processes and estimating its low temperature limit, i.e., the temperature below which amorphous drugs would remain stable. It can also provide comparative estimates of low temperature stability of amorphous drugs in terms of the activation energies of the β-relaxation.     

The Effects of Absorbed Water on the Properties of Amorphous Mixtures Containing Sucrose

Purpose. To measure the water vapor absorption behavior of sucrose-poly(vinyl pyrrolidone) (PVP) and sucrose-poly(vinyl pyrrolidone co-vinyl acetate) (PVP/VA) mixtures, prepared as amorphous solid solutions and as physical mixtures, and the effect of absorbed water on the amorphous properties, i.e., crystallization and glass transition temperature, T_g, of these systems. Methods. Mixtures of sucrose and polymer were prepared by co-lyophilization of aqueous sucrose-polymer solutions and by physically mixing amorphous sucrose and polymer. Absorption isotherms for the individual components and their mixtures were determined gravimetrically at 30°C as a function of relative humidity. Following the absorption experiments, mixtures were analyzed for evidence of crystallization using X-ray powder diffraction. For co-lyophilized mixtures showing no evidence of crystalline sucrose, T_g was determined as a function of water content using differential scanning calorimetry. Results. The absorption of water vapor was the same for co-lyophilized and physically mixed samples under the same conditions and equal to the weighted sums of the individual isotherms where no sucrose crystallization was observed. The crystallization of sucrose in the mixtures was reduced relative to sucrose alone only when sucrose was molecularly dispersed (co-lyophilized) with the polymers. In particular, when co-lyophilized with sucrose at a concentration of 50%, PVP was able to maintain sucrose in the amorphous state for up to three months, even when the T_g was reduced well below the storage temperature by the absorbed water. Conclusions. The water vapor absorption isotherms for co-lyophilized and physically mixed amorphous sucrose-PVP and sucrose-PVP/VA mixtures at 30°C are similar despite interactions between sugar and polymer which are formed when the components are molecularly dispersed with one another. In the presence of absorbed water the crystallization of sucrose was reduced only by the formation of a solid-solution, with PVP having a much more pronounced effect than PVP/VA. The effectiveness of PVP in preventing sucrose crystallization when significant levels of absorbed water are present was attributed to the molecular interactions between sucrose, PVP and water.     

Molecular Dynamics and Physical Stability of Pharmaceutical Co-amorphous Systems: Correlation Between Structural Relaxation Times Measured by Kohlrausch-Williams-Watts With the Width of the Glass Transition Temperature (ΔTg) and the Onset of Crystallization

The study aims to characterize the structural relaxation times of quench-cooled co-amorphous systems using Kohlrausch-Williams-Watts (KWW) and to correlate the relaxation data with the onset of crystallization. Comparison was also made between the relaxation times obtained by KWW and the width of glass transition temperature (ΔT_g) methods (simple and quick). Differential scanning calorimetry, Fourier-transformed infrared spectroscopy, and polarized light microscopy were used to characterize the systems. Results showed that co-amorphous systems yielded a single T_g and ΔC_p, suggesting the binary mixtures exist as a single amorphous phase. A narrow step change at T_g indicates the systems were fragile glasses. In co-amorphous nap-indo and para-indo, experimental T_gs were in good agreement with the predicted T_g. However, the T_g of co-amorphous nap-cim and indo-cim were 20°C higher than the predicted T_g, possibly due to stronger molecular interactions. Structural relaxation times below the experimental T_g were successfully characterized using the KWW and ΔT_g methods. The comparison plot showed that KWW data are directly proportional to the ½ power of ΔT_g data, after adjusting for a small offset. A moderate positive correlation was observed between the onset of crystallization and the KWW data. Structural relaxation times may be useful predictor of physical stability of co-amorphous systems.     

Influence of the Active Pharmaceutical Ingredient Concentration on the Physical State of Mannitol—Implications in Freeze-Drying

Purpose The aim of this study was to investigate the effect of the concentration of the active pharmaceutical ingredient on the physical state of mannitol in frozen aqueous systems. Methods A human monoclonal antibody was used as the model protein. Mannitol and sucrose were used as the bulking agent and the lyoprotectant, respectively. The thermal behavior of frozen mannitol–sucrose solutions during and after annealing, in the absence and presence of the protein, were characterized by low-temperature powder X-ray diffractometry and differential scanning calorimetry. The influence of the protein on the crystallization behavior of mannitol was also evaluated. Results The excipient concentration had a pronounced effect on the glass transition temperature of maximally freeze-concentrated amorphous phase (T_g′). At fixed excipient compositions, the protein had no effect on the T_g′ if the protein concentration was ≤20 mg/ml. However, at higher protein concentrations, there was a marked increase in T_g′ as a function of protein concentration. The inhibitory effect of the protein on mannitol crystallization was concentration dependent and was directly evident from X-ray diffractometry experiments. Annealing facilitated both mannitol nucleation and crystal growth even in the presence of the protein. Conclusions The ratio of mannitol to sucrose and the protein concentration have an impact on the T_g′ and may therefore influence the primary drying temperature. The protein inhibits both the nucleation and growth of mannitol crystals and this effect seems to be concentration dependent. The presence of the protein and the protein concentration dictate the processing conditions, i.e., annealing time, annealing temperature, and primary drying temperature.     

Evaluation of the Physical Stability of Freeze-Dried Sucrose-Containing Formulations by Differential Scanning Calorimetry

Freeze-dried samples of sucrose with buffer salts, amino acids, or dextran have been analyzed with differential scanning calorimetry (DSC) to evaluate the use of DSC thermograms in predicting the physical storage stability. The glass transition temperature, T _g, of the amorphous cake, crystallization, and melting of sucrose are observed with DSC. T _g appeared to be an important characteristic of the physical stability of the amorphous freeze-dried cake. A storage temperature above T _g results in collapse or shrinkage of the cake, which for a sucrose-based formulation, may be accompanied by crystallization of the sucrose. The T _g of the amorphous sucrose is influenced by other components present in the cake. Dextran-40 raised T _g, while the addition of glycine to the formulation lowered T _g. The residual moisture content strongly influences T _g, since water acts as a plasticizer of the system; the higher the moisture content, the lower the T _g and the less physically stable the freeze-dried cake. Crystallization of amorphous sucrose is shown to be inhibited by high molecular weight components or ionic compounds. DSC analysis of freeze-dried cakes proved to be a powerful tool in formulation studies.     

Time-Dependence of Molecular Mobility during Structural Relaxation and its Impact on Organic Amorphous Solids: An Investigation Based on a Calorimetric Approach

Purpose To develop a calorimetry-based model for estimating the time-dependence of molecular mobility during the isothermal relaxation of amorphous organic compounds below their glass transition temperature (T _g).Methods The time-dependent enthalpy relaxation times of amorphous sorbitol, indomethacin, trehalose and sucrose were estimated based on the nonlinear Adam‐Gibbs equation. Fragility was determined from the scanning rate dependence of T _g. Time evolution of the fictive temperature was determined from T _g, the heat capacity of the amorphous and crystalline forms, and from the enthalpy relaxation data.Results Relaxation time changes significantly upon annealing for all compounds studied. The magnitude of the increase in relaxation time does not depend on any one parameter but on four parameters: T _g, fragility, and the crystal–liquid and glass–liquid heat capacity differences. The obtained mobility data for indomethacin and sucrose, both stored at T _g−16 K, correlated much better with their different crystallization tendencies than did the Kohlrausch‐Williams‐Watts (KWW) equation.Conclusions The observed changes in relaxation time help explain and address the limitations of the KWW approach. Due consideration of the time-dependence of molecular mobility upon storage is a key element for improving the understanding necessary for stabilizing amorphous formulations.     

Temperature- and Moisture-Induced Crystallization of Amorphous Lactose in Composite Particles with Sodium Alginate Prepared by Spray-Drying

The purpose of this study was to investigate the temperature- and moisture-induced crystallization of amorphous lactose in the composite particles prepared by spray-drying an aqueous solution of crystalline lactose and sodium alginate. The temperature-induced crystallization of amorphous lactose in the composite particles was suppressed by increasing the amount of sodium alginate in the particles. The stabilizing effect of sodium alginate on amorphous lactose in the composite particles was greater than that in physical mixtures having the same formulating ratios. The improved stability of amorphous lactose in the composite particles was attributed to an increase in the glass transition temperature (T_g) of the mixture. Moisture-induced crystallization of amorphous lactose was also retarded by increasing the amount of sodium alginate in composite particles. Although the T_g of the mixture was reduced by increasing the water content of the particles, the values were higher than that of 100% amorphous lactose when particles of the same water content were compared. The change in the T_g of the composite particles with increasing water content was interpreted as involving three components of the Gordon–Taylor equation. In the amorphous lactose–sodium alginate systems, the T_g values of the composite particles containing sodium alginate were higher than the theoretical line predicted by two components of the Gordon–Taylor equation. These results suggested that there was a specific interaction between the sodium alginate and lactose molecules. This specific interaction was suggested by the fact that only very little amorphous lactose was measured in the spray-dried composite particles stored under humid conditions using differential scanning calorimetry. This molecular interaction may also be partly responsible for the suppression of both the temperature- and moisture-induced crystallization of amorphous lactose in the composite particles.     

Molecular Relaxation Behavior and Isothermal Crystallization above Glass Transition Temperature of Amorphous Hesperetin

The purpose of this paper was to investigate the relaxation behavior of amorphous hesperetin (HRN), using dielectric spectroscopy, and assessment of its crystallization kinetics above glass transition temperature (T_g). Amorphous HRN exhibited both local (β-) and global (α-) relaxations. β-Relaxation was observed below T_g, whereas α-relaxation prominently emerged above T_g. β-Relaxation was found to be of Johari–Goldstein type and was correlated with α-process by coupling model. Secondly, isothermal crystallization experiments were performed at 363 K (T_g+ 16.5 K), 373 K (T_g+ 26.5 K), and 383 K (T_g+ 36.5 K). The kinetics of crystallization, obtained from the normalized dielectric strength, was modeled using the Avrami model. Havriliak–Negami (HN) shape parameters, α_HNand α_HN.β_HN, were analyzed during the course of crystallization to understand the dynamics of amorphous phase during the emergence of crystallites. HN shape parameters indicated that long range (α-like) were motions affected to a greater extent than short range (β-like) motions during isothermal crystallization studies at all temperature conditions. The variable behavior of α-like motions at different isothermal crystallization temperatures was attributed to evolving crystallites with time and increase in electrical conductivity with temperature. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:167–178, 2014     

A Calorimetric Method to Estimate Molecular Mobility of Amorphous Solids at Relatively Low Temperatures

Purpose To present a calorimetry-based approach for estimating the initial (at the onset of annealing) relaxation time (τ ⁰) of organic amorphous solids at relatively low temperatures, and to assess the temperature where molecular mobility of the amorphous drug is reduced to a level comparable with the desired shelf-life of the product.Materials and Methods Values of τ ⁰ for six amorphous pharmaceutical compounds were estimated based on the nonlinear Adam–Gibbs equation. Fragility was determined from the scanning rate-dependence of the glass transition temperature (T _g). The initial enthalpic and entropic fictive temperatures were obtained from the T _g and the heat capacities (C _p) of the amorphous and crystalline forms.Results At a relatively low temperature (∼40°C or more below T _g), τ ⁰ for the different compounds varies by over an order of magnitude. For some materials, the practical storage temperature at T _g − 50 K was found to be still too high to ensure long-term stability. The estimated τ ⁰ is highly sensitive to the fragility of the material and the C _p of the crystalline and amorphous forms. Materials with high fragility or greater C _p differences between crystalline and amorphous forms tend to have longer τ ⁰.Conclusions The proposed method can be used to estimate molecular mobility at relatively low temperatures without having to conduct enthalpy recovery experiments. An accurate τ ⁰ determination from this method relies on faithful fragility measurements.     

Effect of Moisture on the Stability of a Lyophilized Humanized Monoclonal Antibody Formulation

Purpose. To determine the effect of moisture and the role of the glass transition temperature (T_g) on the stability of a high concentration, lyophilized, monoclonal antibody. Methods. A humanized monoclonal antibody was lyophilized in a sucrose/histidine/polysorbate 20 formulation. Residual moistures were from 1 to 8%. T_g values were measured by modulated DSC. Vials were stored at temperatures from 5 to 50°C for 6 or 12 months. Aggregation was monitored by size exclusion chromatography and Asp isomerization by hydrophobic interaction chromatography. Changes in secondary structure were monitored by Fourier transform infrared (FTIR). Results. T_g values varied from 80°C at 1% moisture to 25°C at 8% moisture. There was no cake collapse and were no differences in the secondary structure by FTIR. All formulations were stable at 5°C. High moisture cakes had higher aggregation rates than drier samples if stored above their T_g values. Intermediate moisture vials were more stable to aggregation than dry vials. High moisture samples had increased rates of Asp isomerization at elevated temperatures both above and below their T_g values. Chemical and physical degradation pathways followed Arrhenius kinetics during storage in the glassy state. Only Asp isomerization followed the Arrhenius model above the T_g value. Both chemical and physical stability at T T_g were fitted to Williams-Landel-Ferry (WLF) kinetics. The WLF constants were dependent on the nature of the degradation system and were not characteristic of the solid system. Conclusion. High moisture levels decreased chemical stability of the formulation regardless of whether the protein was in a glassy or rubbery state. In contrast, physical stability was not compromised, and may even be enhanced, by increasing residual moisture if storage is below the T_g value.     

Anti-plasticizing Effect of Amorphous Indomethacin Induced by Specific Intermolecular Interactions with PVA Copolymer

The mechanism of how poly(vinyl alcohol-co-acrylic acid-co-methyl methacrylate) (PVA copolymer) stabilizes an amorphous drug was investigated. Solid dispersions of PVA copolymer, poly(vinyl pyrrolidone) (PVP), and poly(vinyl pyrrolidone-co-vinyl acetate) (PVPVA) with indomethacin (IMC) were prepared. The glass transition temperature (T_g)-proportion profiles were evaluated by differential scanning calorimetry (DSC). General T_g profiles decreasing with the IMC ratio were observed for IMC–PVP and IMC–PVPVA samples. An interesting antiplasticizing effect of IMC on PVA copolymer was observed; T_g increased up to 20% IMC ratio. Further addition of IMC caused moderate reduction with positive deviation from theoretical values. Specific hydrophilic and hydrophobic interactions between IMC and PVA copolymer were revealed by infrared spectra. The indole amide of IMC played an important role in hydrogen bonding with PVA copolymer, but not with PVP and PVPVA. X-ray diffraction findings and the endotherm on DSC profiles suggested that PVA copolymer could form a semicrystalline structure and a possibility of correlation of the crystallographic nature with its low hygroscopicity was suggested. PVA copolymer was able to prevent crystallization of amorphous IMC through both low hygroscopicity and the formation of a specific intermolecular interaction compared with that with PVP and PVPVA.     

Thermal insight of mechanically activated bile acid powders

Mechanical activation of pharmaceutical materials presents an important but poorly understood phenomenon of milled molecular crystals. In this work, a strategy was followed in an effort to understand this phenomenon, cryo-milled of both crystalline and amorphous counterpart of bile acids materials were characterized by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC). The XRPD results for the 30-min milled crystalline powders displayed a characteristic amorphous halo patterns for all compounds tested. The DSC thermograms exhibited the typical glass transition temperatures (T_g) associated with amorphous but only for two materials. For the remaining four milled compounds, a rather interesting behavior was manifested through a characteristic exothermal bimodal peak. The findings seemed to suggest that the occurrence of this event was not related to the (T_g), but likely to the melting temperature (T_m). The DSC results for the melt-quenched (amorphous) ursodeoxycholic acid after cryo-milling revealed that the material crystallized after the influence of the mechanical stress, and a bimodal peak was also observed similar to that of the cryo-milled crystalline material. It is contemplated that the response of the physical instability of the disordered phase could be explained either by the result of surface crystallization kinetics which is different from that of the bulk crystallization, or by the creation of supersaturated dislocated crystal prior to amorphization.     

Ultrasound Assisted Engineering of Lactose Crystals

Purpose  To engineer lactose crystals of desired size, shape, surface and particle size distribution (PSD) as a carrier for dry powder inhalers (DPI) by ultrasound assisted in-situ seeding. Methods  Lactose crystals were obtained from solution by ultrasound assisted in-situ seeding, followed by growth in viscous glycerin solution. The crystals were characterized for physical properties and 63–90 μm size fractions of different batches were mixed with salbutamol sulphate (SS) and compared for in-vitro deposition. Results  Cooling crystallization with stirring for 10–20 h resulted in crystals with wide PSD and varied shape. Application of ultrasound resulted in rapid and complete crystallization in 5 min with rod-shaped fine crystals (15–30 μm) and narrow PSD. In-situ seeded batches yielded micro-fine rod-shaped seed crystals. Seeding followed by growth in glycerin showed desirable size, high elongation ratio, smooth surface and narrow PSD, while growth under stirring showed high elongation ratio with rough surface. Crystals grown in glycerin showed highest dispersibility and fine particle fraction (FPF) of SS. Conclusions  Ultrasound assisted in-situ seeding, followed by ordered growth in glycerin offers rapid technique for separation of nuclei induction from crystal growth yielding desirable characteristics for better dispersion and in-vitro deposition when employed as DPI carrier.     

Significance of Local Mobility in Aggregation of β-Galactosidase Lyophilized with Trehalose,Sucrose or Stachyose

Purpose The purpose of this study is to compare the effects of global mobility, as reflected by glass transition temperature (T_g) and local mobility, as reflected by rotating-frame spin-lattice relaxation time (T_1ρ) on aggregation during storage of lyophilized β-galactosidase (β-GA). Materials and Methods The storage stability of β-GA lyophilized with sucrose, trehalose or stachyose was investigated at 12% relative humidity and various temperatures (40–90°C). β-GA aggregation was monitored by size exclusion chromatography (SEC). Furthermore, the T_1ρ of the β-GA carbonyl carbon was measured by ¹³C solid-state NMR, and T_g was measured by modulated temperature differential scanning calorimetry. Changes in protein structure during freeze drying were measured by solid-state FT-IR. Results The aggregation rate of β-GA in lyophilized formulations exhibited a change in slope at around T_g, indicating the effect of molecular mobility on the aggregation rate. Although the T_g rank order of β-GA formulations was sucrose < trehalose < stachyose, the rank order of β-GA aggregation rate at temperatures below and above T_g was also sucrose < trehalose < stachyose, thus suggesting that β-GA aggregation rate is not related to (T-T_g). The local mobility of β-GA, as determined by the T_1ρ of the β-GA carbonyl carbon, was more markedly decreased by the addition of sucrose than by the addition of stachyose. The effect of trehalose on T_1ρ was intermediate when compared to those for sucrose and stachyose. These findings suggest that β-GA aggregation rate is primarily related to local mobility. Significant differences in the second derivative FT-IR spectra were not observed between the excipients, and the differences in β-GA aggregation rate observed between the excipients could not be attributed to differences in protein secondary structure. Conclusions The aggregation rate of β-GA in lyophilized formulations unexpectedly correlated with the local mobility of β-GA, as indicated by T_1ρ, rather than with (T-T_g). Sucrose exhibited the most intense stabilizing effect due to the most intense ability to inhibit local protein mobility during storage.     

Thermal Behavior of Poly Hydroxy Ethyl Methacrylate (pHEMA) Hydrogels

The freezing and melting behavior of water in poly hydroxy ethyl methacrylate (pHEMA) hydrogels of different cross-linker and water contents was investigated in relation to the glass transition temperature (T _g) of the gels. After prolonged cooling at –15°C a constant amount of 1.7 mol water per monomeric unit did not freeze, regardless of both the cross-linker and the water content of the gels. At this water content and temperature, pHEMA gels were below their T _g, and the water molecules were prevented from diffusing to the ice crystals formed in the gel. Therefore, the inability of part of the water in pHEMA gels to freeze is not a thermodynamic phenomenon but is caused by kinetic factors.