The Effect of Stabilizers and Denaturants on the Cold Denaturation Temperatures of Proteins and Implications for Freeze-Drying

Purpose The aim of the study is to investigate the effects of stabilizers and denaturants on the thermal and cold denaturation temperatures of selected proteins in systems of interest to freeze-drying.Methods β-Lactoglobulin and phosphoglycerate kinase (PGK) were chosen as model proteins. Protein thermal and cold denaturation temperatures were determined by both conventional and modulated differential scanning calorimetry and verified by tryptophan emission spectroscopy in selected systems.Results The cold denaturation of β-lactoglobulin was reversible, whereas the thermal denaturation was only reversible at high scanning rate (10°C/min). The cold denaturation temperatures of β-lactoglobulin decreased with an increase in protein concentration (self-stabilization). The cold denaturation temperature increased with increases in pH (from pH 2 to 7) with about 4.6°C increase per unit pH change. All stabilizers studied (i.e., sucrose, trehalose and glycerol) increased the thermal denaturation temperature of the proteins studied and decreased the cold denaturation temperature. The effect of sucrose in decreasing the PGK cold denaturation temperature [40°C per molar concentration increase (40°C/M)] was of the same magnitude as for β-lactoglobulin (36°C/M). The effect of stabilizers on cold denaturation temperatures is much greater than the effect on thermal denaturation temperatures. With sucrose, the β-lactoglobulin thermal denaturation temperature increases only about 5°C from 0 to 2.7 M, whereas the decrease in cold denaturation temperature was more than 35°C even at sucrose concentrationsas low as 0.9 M. Denaturants (urea and guanidine hydrochloride) increased the cold denaturationtemperatures of proteins and thereby destabilized protein; the magnitudes were 9°C/M (urea on T_cd of β-lactoglobulin) and 65°C/M (guanidine hydrochloride on PGK) compared with literature data of 16°C/M (guanidine hydrochloride on β-lactoglobulin). The cold denaturation temperatures of β-lactoglobulinand PGK extrapolated to zero concentration of denaturants were −14 and −26°C, respectively.Conclusions The protein cold denaturation temperature was pH-, protein concentration-, and additive-dependent. Stabilizers, such as sugars and/or polyols, can stabilize both protein thermal and cold denaturation, whereas the denaturants destabilize protein cold denaturation. The stabilization effect on protein cold denaturation is much larger than on thermal denaturation, a result of great importance in protein freeze-drying.     

Using dextran of different molecular weights to achieve faster freeze-drying and improved storage stability of lactate dehydrogenase

Freeze-drying of protein formulations is frequently used to maintain protein activity during storage. The freeze-drying process usually requires long primary drying times because the highest acceptable drying temperature to obtain acceptable products is dependent on the glass transition temperature of the maximally freeze-concentrated solution (T_g′). On the other hand, retaining protein activity during storage is related to the glass transition temperature (T_g) of the final freeze-dried product. In this study, dextrans with different molecular weight (1 and 40?kDa) and mixtures thereof at the ratio 3:1, 1:1, and 1:3 (w/w) were used as cryo-/lyoprotectant and their impact on the stability of the model protein lactate dehydrogenase (LDH) was investigated at elevated temperatures (40?°C and 60?°C). The dextran formulations were then compared to formulations containing sucrose as cryo-/lyoprotectant. Because of the higher T_g′ values of the dextrans, the primary drying times could be reduced compared to freeze-drying with sucrose. Similarly, the higher T_g and T_g′ of dextrans relative to sucrose led to benefits during storage which was shown through improved protection of LDH activity.     

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.     

Stabilizing Effect of Four Types of Disaccharide on the Enzymatic Activity of Freeze-dried Lactate Dehydrogenase: Step by Step Evaluation from Freezing to Storage

Purpose In order to understand the stabilizing effects of disaccharides on freeze-dried proteins, the enzymatic activity of lactate dehydrogenase (LDH) formulations containing four types of disaccharide (trehalose, sucrose, maltose, and lactose) at two relative humidity (RH) levels (about 0 and 32.8%) was investigated after three processes: freeze-thawing, freeze-drying, and storage at three temperatures (20, 40, and 60°C) above and/or below the glass transition temperature (T _g). Materials and Methods The enzymatic activity was determined from the absorbance at 340 nm, and T _g of the samples was investigated by differential scanning calorimetry. Results At each RH condition, T _g values of sucrose formulations were lower than those of other formulations. Although effects of the disaccharides on the process stability of LDH were comparable, storage stability was dependent on the type of disaccharide. All the formulations were destabilized significantly during storage at temperature above T _g. During storage at temperature below T _g, the LDH activity decreased with increases in the storage temperature and moisture. Maltose and lactose formulations showed significant destabilization with the change of color to browning. Conclusions Taking the storage stability of freeze-dried proteins under the various conditions (temperature and RH) into consideration, trehalose is better suited as the stabilizer than other disaccharides.     

Effect of Sucrose/Raffinose Mass Ratios on the Stability of Co-Lyophilized Protein During Storage Above the Tg

Purpose. To examine the potential of raffinose as an excipient in stabilizing protein and to study the effect of sucrose/raffinose mass ratios on the stability of co-lyophilized protein and amorphous solids during storage at an elevated temperature. Methods. Glucose-6-phosphate dehydrogenase (G6PDH) was co-lyophilized with sucrose and raffinose mixed at different mass ratios. The activity of dried G6PDH was monitored during storage at 44°C. Thermal properties of sucrose/raffinose matrices were determined by differential scanning calorimetry (DSC). Results. Mass ratios of sucrose to raffinose did not affect the recovery of G6PDH activity after freeze-drying, but significantly affected the stability of freeze-dried G6PDH during storage. The sucrose-alone formulation offered the best enzyme stabilization during storage. With increasing fraction of raffinose, the G6PDH stability decreased, sugar crystallization inhibited, and crystal-melting temperature increased. Conclusions. Despite the higher T_g of the formulations with higher fraction of raffinose, they provided less protection for G6PDH than did sucrose alone during storage. Our data do not support the prediction from recent thermophysical studies that raffinose should be superior to sucrose and trehalose as a potential excipient or stabilizer.     

Effect of Glass Transition Temperature on the Stability of Lyophilized Formulations Containing a Chimeric Therapeutic Monoclonal Antibody

Purpose. The purpose of this study is to highlight the importance of knowing the glass transition temperature, T_g, of a lyophilized amorphous solid composed primarily of a sugar and a protein in the interpretation of accelerated stability data. Methods. Glass transition temperatures were measured using DSC and dielectric relaxation spectroscopy. Aggregation of protein in the solid state was monitored using size-exclusion chromatography. Results. Sucrose formulation (T_g ~ 59°C) when stored at 60°C was found to undergo significant aggregation, while the trehalose formulation (T_g ~ 80°C) was stable at 60°C. The instability observed with sucrose formulation at 60°C can be attributed to its T_g (~59°C) being close to the testing temperature. Increase in the protein/sugar ratio was found to increase the T_gs of the formulations containing sucrose or trehalose, but to different degrees. Conclusions. Since the formulations exist in glassy state during their shelf-life, accelerated stability data generated in the glassy state (40°C) is perhaps a better predictor of the relative stability of formulations than the data generated at a higher temperature (60°C) where one formulation is in the glassy state while the other is near or above its T_g.     

Freeze-Concentration Separates Proteins and Polymer Excipients Into Different Amorphous Phases

Purpose. To study the miscibility of proteins and polymer excipients in frozen solutions and freeze-dried solids as protein formulation models. Methods. Thermal profiles of frozen solutions and freeze-dried solids containing various proteins (lysozyme, ovalbumin, BSA), nonionic polymers (Ficoll, polyvinylpyrrolidone [PVP]), and salts were analyzed by differential scanning calorimetry (DSC). The polymer miscibility was determined from the glass transition temperature of maximally freeze-concentrated solute (T_g) and the glass transition temperature of freeze-dried solid (T_g). Results. Frozen Ficoll or PVP 40k solutions showed T_g at –22°C, while protein solutions did not show an apparent T_g. All the protein and nonionic polymer combinations (5% w/w, each) were miscible in frozen solutions and presented single T_gs that rose with increases in the protein ratio. Various salts concentration-dependently lowered the single T_gs of the proteins and Ficoll combinations maintaining the mixed amorphous phase. In contrast, some salts induced the separation of the proteins and PVP combinations into protein-rich and PVP-rich phases among ice crystals. The T_gs of these polymer combinations were jump-shifted to PVP's intrinsic T_g at certain salt concentrations. Freeze-dried solids showed varied polymer miscibilities identical to those in frozen solutions. Conclusions. Freeze-concentration separates some combinations of proteins and nonionic polymers into different amorphous phases in a frozen solution. Controlling the polymer miscibility is important in designing protein formulations.     

Development of an Efficient Single-Step Freeze-Drying Cycle for Protein Formulations

Purpose. An efficient freeze-drying cycle for recombinant human interleukin-1 receptor antagonist (rhIL-lra) formulations, which contained glycine and sucrose as excipients, was developed. Methods. Development was based on characterizing the frozen formulations by thermal analysis and by examining the effect of various lyophilization process parameters on the sublimation rate of ice. Results. Thermal analysis showed that the metastable glass of glycine in frozen formulation could be devitrified by slowly warming the frozen product to –15°C. During drying, the sublimation rate of ice was increased as a linear function of the difference between the vapor pressure of ice at the product temperature (P _O) and the chamber pressure (P _C). Therefore, the product temperature (T_p) was maintained as high as possible at temperatures below Tg of the formulation, in order to maximize the P _O without allowing the collapse of cake. Although various combinations of shelf temperatures and chamber pressures could be used to obtain the same T_p, the combination of higher shelf temperature and lower chamber pressure was used to maximize sublimation rate. Conclusions. A single-step drying cycle was developed to take advantage of these observations. The shelf temperature was set for the secondary drying and the product temperature during primary drying was maintained below T_g by adjusting the chamber pressure. As the sublimation completed, the product temperature increased naturally to the shelf temperature for the secondary drying. This process resulted in successful drying of 1 ml of rhIL-lra formulation to 0.4% moisture content within 6 hours.     

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.     

Characterization of Murine Monoclonal Antibody to Tumor Necrosis Factor (TNF-MAb) Formulation for Freeze-Drying Cycle Development

Purpose. This study was designed to characterize the formulation of protein pharmaceuticals for freeze-drying cycle development. Thermal properties of a protein formulation in a freezing temperature range are important in the development of freezing and primary drying phases. Moisture sorption properties and the relationship between moisture and stability are the bases for the design of the secondary drying phase. Methods. We have characterized the formulation of TNF-MAb for the purpose of freeze-drying cycle development. The methods include: DTA with ER probes, freeze-drying microscopy, isothermal water adsorption, and moisture optimization.Results. The DTA/ER work demonstrated the tendency to noneutectic freezing for the TNF-MAb formulation at cooling rates of –1 to –3°C/min. The probability of glycine crystallization during freezing was quite low. A special treatment, either a high subzero temperature holding or annealing could promote the maximum crystallization of glycine, which could dramatically increase the T_g' of the remaining solution. The freeze-drying microscopy further indicated that, after the product was annealed, the cake structure was fully maintained at a T_p below –25°C during primary drying. The moisture optimization study demonstrated that a drier TNF-MAb product had better stability. Conclusions. An annealing treatment should be implemented in the freezing phase in order for TNF-MAb to be dried at a higher product temperature during primary drying. A secondary drying phase at an elevated temperature was necessary in order to achieve optimum moisture content in the final product.     

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

Purpose. The effect of the molecular weight of dextran on the molecular mobility and protein stability of freeze-dried serum -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 (T_mc), at which dextran protons started to exhibit a Lorentzian relaxation decay due to higher mobility in addition to a Gaussian relaxation decay. T_mc increased as the molecular weight of dextran increased. The proportion of dextran protons which exhibited the higher mobility relaxation process (P_hm) at temperatures above T_mc 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 T_mc, and denaturation in the microscopically liquidized state decreased as P_hm 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 T_mc and P_hm. These parameters appear to be useful in preformulation and stability prediction of freeze-dried formulations.     

Reversibility of Heat-Induced Denaturation of the Recombinant Human Megakaryocyte Growth and Development Factor

Purpose. The present study was performed to examine the effect of solution conditions on the reversibility of the thermal denaturation of megakaryocyte growth and development factor (rHuMGDF). Methods. Changes in the far U V CD spectra of rHuMGDF with temperature were used to monitor the thermal denaturation of the protein, and the recovery of folded protein following a return to room temperature. The effect of protein concentration, scan rate, and buffer composition on thermal denaturation and on the reversibility were determined. Surface tension measurements were used to determine the effect of this unfolding reaction on the surface adsorption of the protein. Sedimentation velocity was used to assess recovery of native monomer and the size of soluble aggregates. In addition, monomeric protein remaining in solution after incubation at 37°C for 2 weeks in either 10 mM imidazole of 10 mM phosphate was determined. Results. In phosphate buffer the rHuMGDF irreversibly precipitates upon unfolding under all the conditions examined. In imidazole the unfolding is at least partially reversible, with no visible precipitate seen; the degree of reversibility increased by lowering both protein and salt concentrations, and the amount of time spent at elevated temperature. In order to compare thermal unfolding occuring with different degrees of reversibility, the melting temperature was defined as the temperature at which melting begins. The melting temperature itself is relatively independent of the buffer composition, or experimental conditions. At low protein concentrations the protein stabilizer sucrose had a marginal effect on the thermal transition of rHuMGDF, while at protein concentrations of about 2 mg/ml the inclusion of sucrose increased the apparent melting temperature by about 4°C, to that seen at low protein concentrations, but had little effect on the reversibility of denaturation. Inclusion of 1 or 2 M urea did not affect the reaction. Surface tension measurements of rHuMGDF solutions showed little difference before and after melting, and in the presence or absence of sucrose. When unfolding is irreversible, the MGDF appears to form soluble aggregates of tetramers to 14-mers, while under reversible conditions native monomer is recovered. More monomeric MGDF remained in solution following storage for 2 weeks at 37°C in imidazole than in phosphate, in both the presence and absence of sucrose. Conclusions. These results can be explained by assuming that thermal denaturation proceeds as a two-step reaction, the first step being the equilibrium between folded and unfolded states, while the second step is a slow irreversible aggregation. The different buffer systems affect the rate of the aggregation step, but not the intrinsic thermal stability nor the rate of the unfolding step.     

Effects of Additives on Heat Denaturation of rhDNase in Solutions

Purpose. To study the thermal stability of recombinant human deoxyribonuclease I (rhDNase) in aqueous solutions. Methods. Differential scanning calorimetry (DSC) was used to measure the denaturation or melting temperature (T_m) and enthalpy (H_m) of rhDNase. The effects of denaturants (guanidine HC1 and urea) and additives (mainly divalent cations and disaccharides) were investigated at pH 6–7. Results. The T_m and H_m of rhDNase in pure water were measured as 67.4 °C and 18.0 J/g respectively, values typical of globular proteins. The melting peak disappeared on re-running the sample after cooling to room temperature, indicating that the thermal denaturation was irreversible. The latter was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that during heat denaturation, rhDNase formed soluble high molecular weight aggregates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca²⁺ stabilizes rhDNase against thermal denaturation and elevates T_m and H_m while Mg²⁺, Mn²⁺ and Zn²⁺ destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T_m and H_m. All destabilization of rhDNase can be prevented by adding Ca²⁺ to the solutions. Conclusions. CaCl₂ and sugars were found to stabilize rhDNase against thermal denaturation while divalent cations, urea and guanidine HC1 destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca²⁺ the protective effect is believed to be due to an ordering of the rhDNase structure in its native state, and by prevention of breaking of a disulfide bridge, thus making it less susceptible to unfold under thermal stress.     

Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase.

During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate significantly more inactivation and secondary structural perturbation of monomeric and tetrameric beta-galactosidase (beta-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the significant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer (1). In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric beta-gal in either NaP or KP buffer resulted in significant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilization-induced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native protein in the dried state as well as the complete recovery of active beta-gal on reconstitution. These results indicate that addition of an effective stabilizer, such as sucrose, may minimize protein denaturation during freeze-drying in phosphate buffers, even if there are large-scale changes in solution pH during freezing.     

A Central Composite Design to Investigate the Thermal Stabilization of Lysozyme

Purpose. The formulation and processing of protein drugs requires the stabilization of the native, biologically active structure. Our aim was to investigate the thermal stability of a model protein, lysozyme, in the presence of two model excipients, sucrose and hydroxypropyl--cyclodextrin (HP--CD). Methods. We used high sensitivity differential scanning calorimetry (HSDSC) in combination with a central composite design (CCD). As indicators of protein thermal stability, the measured responses were the unfolding transition temperature (T_m), the onset temperature of the denaturation (T₀), and the extrapolated onset temperature (T_o,e). Results. A highly significant (F probability <0.001) statistical model resulted from analysis of the data. The largest effect was due to pH (over the range 3.2-7.2), and the pH value that maximized T_m was 4.8. Several minor but significant effects were detected that were useful for mechanistic understanding. In particular, the effects of protein concentration and cyclodextrin concentration on T_m and T_o,e were found to be pH-dependent. This was indicative of the partially hydrophilic nature of protein-protein interactions and protein-cyclodextrin interactions, respectively. Conclusions. Response surface methodology (RSM) proved efficient for the modeling and optimization of lysozyme thermal stability as well as for the physical understanding of the protein-sugar-cyclodextrin system in aqueous solution.     

Effects of Solute Miscibility on the Micro- and Macroscopic Structural Integrity of Freeze-Dried Solids

The purpose of this study was to elucidate the effect of solute miscibility in frozen solutions on their micro- and macroscopic structural integrity during freeze-drying. Thermal analysis of frozen solutions containing poly(vinylpyrrolidone) (PVP) and dextran showed single or multiple thermal transitions (T′_g: glass transition temperature of maximally freeze-concentrated solutes) depending on their composition, which indicated varied miscibility of the concentrated noncrystalline polymers. Freeze-drying of the miscible solute systems (e.g., PVP 10,000 and dextran 1060, single T′_g) induced physical collapse during primary drying above the transition temperatures (> T′_g). Phase-separating PVP 29,000 and dextran 35,000 mixtures (two T′_gs) maintained their cylindrical structure following freeze-drying below both of the T′_gs (<?24°C). Primary drying of the dextran-rich systems at temperatures between the two T′_gs (? 20 to ? 14°C) resulted in microscopically disordered “microcollapsed” cake-structure solids. Freeze-drying microscopy (FDM) analysis of the microcollapsing polymer system showed locally disordered solid region at temperatures between the collapse onset (T_c1) and severe structural change (T_c2). The rigid dextran-rich matrix phase should allow microscopic structural change of the higher fluidity PVP-rich phase without loss of the macroscopic cake structure at the temperature range. The results indicated the relevance of physical characterization and process control for appropriate freeze-drying of multicomponent formulations.     

Determination of the Viscosity of an Amorphous Drug Using Thermomechanical Analysis (TMA)

Purpose. To evaluate thermomechanical analysis (TMA) as a technique for determining the viscosity of amorphous pharmaceutical materials. This property of amorphous drugs and excipients is related to their average rate of molecular mobility and thus to their physical and chemical stability. Methods. Indomethacin was selected as a model amorphous drug whose viscosity has previously been reported in the literature. A Seiko TMA 120C thermomechanical analyzer was utilized in isothermal penetration mode to determine the viscosity of the amorphous drug over the maximum possible range of temperatures. Results. Using a cylindrical penetration geometry it was possible to accurately determine the viscosity of amorphous indomethacin samples by TMA over the temperature range from 35 to 75°C. The results were consistent with those reported in the literature using a controlled strain rheometer over the range 44–75°C. The limiting lower experimental temperature for the TMA technique was extended to significantly below the calorimetric glass transition temperature (T_g 42°C), thus allowing a direct experimental determination of the viscosity at T_g to be made. Conclusions. Thermomechanical analysis can be used to accurately determine the viscosity of amorphous pharmaceutical materials at temperatures near and above their calorimetric glass transition temperatures.     

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.     

Molecular mobility in liquid and glassy states of Telmisartan (TEL) studied by Broadband Dielectric Spectroscopy

The molecular relaxation in liquid and glassy states of Telmisartan (TEL) has been studied by Broadband Dielectric Spectroscopy (BDS) covering wide temperature and frequency range. Multiple relaxation processes were observed. Besides the primary α-relaxation, two secondary relaxations β and γ (labelled in order of decreasing time scale) have been reported.Well-separated β-process observed above and below glass transition temperature T_g, has activation energy E_β = 81.8 kJ/mol and was identified as intermolecular Johari–Goldstein (JG) process. The γ-relaxation visible in dielectric loss spectra at very low temperatures is most likely non-JG relaxation. The temperature dependence of the relaxation times of α-process, measured over 11 orders of magnitude, cannot be described by a single Vogel–Fulcher–Tamman–Hesse (VFTH) equation. At temperature T_B = 475.8 K the change in relaxation dynamics occurred, consequently a new set of VFTH parameters was required. From low temperature VFTH fits the glass transition temperature T_g was estimated as T_g = 400.3 K and fragility index m = 87 was calculated. Of particular interest was the time scale of molecular motion below the glass transition temperature. Our observation clearly indicates that the α-relaxation times at room temperature most probably would exceed 3 years and amorphous TEL should maintain physically and chemically stable over prolonged storage time.     

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.