Physical stability of amorphous pharmaceuticals: Importance of configurational thermodynamic quantities and molecular mobility

This work relates the thermodynamic quantities (Gc, Hc, and Sc) and the molecular mobility values (1/tau) of five structurally diverse amorphous compounds to their crystallization behavior. The model compounds included: ritonavir, ABT-229, fenofibrate, sucrose, and acetaminophen. Modulated temperature DSC was used to measure the heat capacities as a function of temperature for the amorphous and crystalline phases of each compound. Knowledge of the heat capacities and fusion data allowed calculation of the configurational thermodynamic quantities and the Kauzmann temperatures (T(K)) using established relationships. The molecular relaxation time constants (tau) were then calculated from the Vogel-Tammann-Fulcher representation of the Adam-Gibbs model. Amorphous samples were heated at 1 K/min and a reduced crystallization temperature, defined as (Tc - Tg)/(Tm-Tg), was used to compare crystallization tendencies. Crystallization was observed for all compounds except ritonavir. The configurational free energy values (Gc) show that thermodynamic driving forces for crystallization follow the order: ritonavir > acetaminophen approximately fenofibrate > sucrose > ABT-229. The entropic barrier to crystallization, which is inversely related to the probability that the molecules are in the proper orientation, followed the order: ritonavir > fenofibrate > ABT-229 > acetaminophen approximately sucrose. Molecular mobility values, which are proportional to molecular collision rates, followed the order: acetaminophen > fenofibrate > sucrose > ABT-229 approximately ritonavir. Crystallization studies under nonisothermal conditions revealed that compounds with the highest entropic barriers and lowest mobilities were most difficult to crystallize, regardless of the thermodynamic driving forces. This investigation demonstrates the importance of both configurational entropy and molecular mobility to understanding the physical stability of amorphous pharmaceuticals.     

The effect of annealing on the stability of amorphous solids: chemical stability of freeze-dried moxalactam

The objective of this study was to investigate the effect of annealing on the chemical stability and calorimetric structural relaxation times of freeze-dried moxalactam. Moxalactam disodium was freeze dried with 12% mannitol and split into several batches after freeze drying. One batch was held as a control while others were subjected to a further heating (annealing) treatment at 60 degrees C, 70 degrees C, and 80 degrees C for different periods of time. Isothermal microcalorimetry studies using thermal activity monitor (TAM) were performed on the freeze-dried samples to measure relaxation times (tau) and stretched exponential values (beta). Modulated DSC experiments were carried out to determine T(g) and DeltaC(P) for moxalactam-12% w/w mannitol systems at various moisture contents to allow extrapolation of these quantities to zero residual moisture. Storage stability studies were performed at 25 degrees C, 40 degrees C and 50 degrees C. Decarboxylated moxalactam and parent contents after various storage times were measured by reverse phase HPLC. Annealing moxalactam-12% mannitol amorphous systems improved chemical stability of moxalactam and reduced molecular mobility, as measured by TAM. Moxalactam-12% w/w mannitol systems annealed at higher temperatures and for longer times had higher tau(beta) values than the "control" sample, with tau(beta) values increasing as annealing temperature increased. Additionally, tau(beta) value increased as annealing time at the same temperature increased. These observations indicated that higher temperature annealing decreased molecular mobility in the glass, as expected. Further, chemical stability improved as annealing temperatures and annealing times increased. For example, the rate of decarboxylation of the sample annealed at 70 degrees C for 8 h was roughly 1.7 times lower than the "control." Note that in spite of degradation during the annealing process, the level of degradation at the end of storage is actually less in the annealed sample than in the control sample; thus, annealing can result in samples having less degradation at the end of a storage period. Chemical stability and relaxation times are correlated, thus indicating that molecular mobility and structural relaxation time are coupled.     

Dynamic heat capacity and relaxation time of ultraviscous melt and glassy acetaminophen

The real and imaginary components, C'(p) and C'(p), of the complex heat capacity, C*(p)=C'(p)-iC"(p) of supercooled, ultraviscous melt of acetaminophen have been measured at different temperatures during cooling through its vitrification range and during heating through its glass-softening range by using a modulation frequency of 3.3 mHz. From these data, the distribution of relaxation time parameter, beta, and a characteristic (calorimetric or configurational) relaxation time, tau(cal), have been determined. A constant value of 0.65 for beta fits the data, and tau(cal) varies with the temperature according to the Vogel-Fulcher-Tammann equation, tau(cal) = 10(-12.95) exp[1813/(T - 240.5)]. This relation differs significantly from the one deduced by others in which the configurational entropy theory was used to deduce tau(cal). The C'(p) and C'(p) values measured during the cooling of its ultraviscous melt and during the heating of its glassy state show a small hysteresis only at low temperatures. These investigations also provide a comparison of calorimetric and dielectric relaxation times in ultraviscous acetaminophen and highlight the role of faster modes of relaxation at low temperatures in organic, molecular glasses that can help in a better understanding of the crystal nucleation process in glasses at T below their T(g).     

Calorimetric relaxation and the glass-liquid temperature range of acetaminophen-nifedipine alloys

Glassy states of nine acetaminophen-nifedipine compositions have been made by slowly supercooling their melts, and calorimetric T(g) and the nonexponential, nonlinear relaxation parameters beta and x that are used in modeling the mobility of a pharmaceutical determined. The T(g)-endotherm's shape varies with the alloy's composition, T(g) increases approximately linearly with the mol% of nifedipine, beta and x increase, and the activation enthalpy Deltah* decreases. At T(g), the relaxation time tau(cal) of acetaminophen, nifedipine, and their alloys differs from 100 s to different extents. The distribution of relaxation times is lesser than that for polymers and other glasses. For a given composition, Deltah*, beta, x, and tau(cal) anomalously depend upon the heating rate, indicating that variation of beta with temperature would not yield better fits for modeling their stability. It is suggested that a pharmaceutical's relaxation is generally influenced by changes in intermolecular hydrogen bonds, chemical short-range order, vibrational frequency, isomerization, and impurity electrolyte dissociation, all of which contribute to the energy change with a distinctive kinetics.     

Temperature- and glass transition temperature-dependence of bimolecular reaction rates in lyophilized formulations described by the Adam-Gibbs-Vogel equation

Bimolecular reaction rates in lyophilized aspirin-sulfadiazine formulations containing poly(vinylpyrrolidone), dextran, and isomalto-oligomers of different molecular weights were determined in the presence of various water contents, and their temperature- and glass transition temperature (Tg)-dependence was compared with that of structural relaxation time (tau calculated according to the Adam-Gibbs-Vogel equation, in order to understand how chemical degradation rates of drugs in lyophilized formulations are affected by molecular mobility. The rate of acetyl transfer in poly(vinylpyrrolidone) K30 and dextran 40k formulations with a constant Tg, observed at various temperatures, exhibited a temperature dependence similar to that of tau at temperatures below Tg. Furthermore, the rates of acetyl transfer and the Maillard reaction in formulations containing alpha-glucose polymers and oligomers increased, as the Tg of formulations decreased, either associated with decreases in molecular weight of excipient or with increases in water content. The observed Tg dependence was similar to that of tau in the range of Tg higher than the experimental temperature. The results suggest a possibility that bimolecular reaction rate at temperatures below Tg can be predicted from that observed at the Tg on the basis of temperature dependence of structural relaxation time in amorphous systems, if the degradation rate is proportional to the diffusion rate of reacting compounds.     

Relationship between the crystallization rates of amorphous nifedipine, phenobarbital, and flopropione, and their molecular mobility as measured by their enthalpy relaxation and (1)H NMR relaxation times

Isothermal crystallization of amorphous nifedipine, phenobarbital, and flopropione was studied at temperatures above and below their glass transition temperatures (T(g)). A sharp decrease in the crystallization rate with decreasing temperature was observed for phenobarbital and flopropione, such that no crystallization was observed at temperatures 20-30 degrees C lower than their T(g) within ordinary experimental time periods. In contrast, the crystallization rate of nifedipine decreased moderately with decreasing temperature, and considerable crystallization was observed at 40 degrees C below its T(g) within 4 months. The molecular mobility of these amorphous drugs was assessed by enthalpy relaxation and (1)H-NMR relaxation measurements. The enthalpy relaxation time of nifedipine was smaller than that of phenobarbital or flopropinone at the same T - T(g) values, suggesting higher molecular mobility of nifedipine. The spin-lattice relaxation time in the rotating frame (T(1rho)) decreased markedly at temperature above T(g). The slope of the Arrhenius type plot of the T(1rho) for nifedipine protons changed at about 10 degrees C below the T(g), whereas the slope for phenobarbital protons became discontinuous at about 10 degrees C above the T(g). Even at temperatures below its T(g), the spin-spin relaxation process of nifedipine could be described by the sum of its Gaussian relaxation, which is characteristic of solid protons, and its Lorentzian relaxation, which is characteristic of protons with higher mobility. In contrast, no Lorentzian relaxation was observed for phenobarbital or flopropione at temperatures below their T(g). These results also suggest that nifedipine has higher molecular mobility than phenobarbital and flopropione at temperatures below T(g). The faster crystallization of nifedipine than that of phenobarbital or flopropione observed at temperatures below its T(g) may be partly ascribed to its higher molecular mobility at these temperatures.     

Investigation of the impact of annealing on global molecular mobility in glasses: optimization for stabilization of amorphous pharmaceuticals

The purpose of this research was to investigate the effect of annealing on the molecular mobility in lyophilized glasses using differential scanning calorimetry (DSC) and isothermal microcalorimetry (IMC) techniques. A second objective that emerged was a systematic study of the unusual pre-T(g) thermal events that were observed during DSC warming scans after annealing. Aspartame lyophilized with three different excipients; sucrose, trehalose and poly vinyl pyrrolidone (PVP) was studied. The aim of this work was to quantify the decrease in mobility in amorphous lyophilized aspartame formulations upon systematic postlyophilization annealing. DSC scans of aspartame:sucrose formulation (T(g) = 73 degrees C) showed the presence of a pre-T(g) endotherm which disappeared upon annealing. Aspartame:trehalose (T(g) = 112 degrees C) and aspartame:PVP (T(g) = 100 degrees C) showed a broad exotherm before T(g) and annealing caused appearance of endothermic peaks before T(g). This work also employed IMC to measure the global molecular mobility represented by structural relaxation time (tau(beta)) in both un-annealed and annealed formulations. The effect of annealing on the enthalpy relaxation of lyophilized glasses, as measured by DSC and IMC, was consistent with the behavior predicted using the Tool-Narayanaswamy-Moynihan (TNM) phenomenology (Luthra et al., 2007, in press). The results show that the systems annealed at T(g) -15 degrees C to T(g) -20 degrees C have the lowest molecular mobility.     

Effect of thermal history on the glassy state of indapamide

The effects of thermal history, e.g. cooling rate, annealing, etc., on the thermal behaviour of indapamide glass were determined by differential scanning calorimetry (DSC). The glass was prepared by heating indapamide crystals (m.p. 162 degrees C) to 180 degrees C, and then cooling the melt to room temperature. The glass transition temperature (Tg) of the material was 98 degrees C. An endotherm, due to thermal relaxation of the glass, was observed in the DSC thermogram when indapamide glass was prepared by slow cooling or was annealed isothermally at a temperature below Tg. Such enthalpy relaxation may be observed during ageing of pharmaceutical glasses and might influence their physico-chemical properties.     

Molecular mobility-based estimation of the crystallization rates of amorphous nifedipine and phenobarbital in poly(vinylpyrrolidone) solid dispersions

The overall crystallization rates and mean relaxation times of amorphous nifedipine and phenobarbital in the presence of poly(vinylpyrrolidone) (PVP) were determined at various temperatures to gain further insight into the effect of molecular mobility on the crystallization rates of amorphous drugs and the possibility of predicting stability from their molecular mobility. Nifedipine-PVP (9:1 w/w) and phenobarbital-PVP (95:5 w/w) solid dispersions were prepared by melting and rapidly cooling mixtures of each drug and PVP. The amount of amorphous nifedipine remaining in the solid dispersion was calculated from the heat of crystallization,which was obtained by differential scanning calorimetry. The amount of amorphous phenobarbital remaining in the solid dispersion was estimated from the change in the heat capacity at its glass transition temperature (T(g)). The time required for the amount of amorphous drug remaining to fall to 90% (t(90)) was calculated from the profile of time versus the amount of amorphous drug remaining. The t(90) values for the solid dispersions studied were 100-1000 times longer than those of pure amorphous drugs when compared at the same temperature. Enthalpy relaxation of the amorphous drugs in the solid dispersions was reduced compared with that in the pure amorphous drugs, indicating that the molecular mobility of the amorphous drugs is reduced in the presence of PVP. The temperature dependence of mean relaxation time (tau) for the nifedipine-PVP solid dispersion was calculated using the Adam-Gibbs-Vogel equation. Parameters D and T(0) in this equation were estimated from the heating rate dependence of T(g). Similar temperature dependence was observed for t(90) and tau values of the solid dispersion, indicating that the information on the temperature dependence of the molecular mobility, along with the crystallization data obtained at around the T(g), are useful for estimating the t(90) of overall crystallization at temperatures below T(g) in the presence of excipients.     

Glass transition-related changes in molecular mobility below glass transition temperature of freeze-dried formulations, as measured by dielectric spectroscopy and solid state nuclear magnetic resonance

The purpose of this study was to explore why changes in the molecular mobility associated with glass transition, the timescale of which is on the order of 100 s, can be detected by measuring the nuclear magnetic resonance relaxation times that reflect molecular motions on the order of 10 kHz and 1 MHz. The molecular motions in freeze-dried dextran 40k, dextran 1k, isomaltotriose (IMT), and alpha-glucose comprising a common unit but with different glass transition temperatures, were investigated by dielectric spectroscopy (DES) in the frequency range of 0.01 Hz to 100 kHz and in the temperature range of -20 degrees to 200 degrees C, in order to compare with the molecular motions reflected in nuclear magnetic resonance relaxation times. The alpha-relaxation process for freeze-dried alpha-glucose was visualized by DES, whereas those for freeze-dried dextran 40k, dextran 1k, and IMT were too slow to be visualized by DES. The latter freeze-dried cakes exhibited quasi-dc polarization because of proton-hopping-like motion rather than alpha-relaxation process. The correlation time (tau(c)) for the backbone carbon of dextran 40k and IMT, calculated from the measured value of spin-lattice relaxation time in the rotating frame, was found to be close to the relaxation time of proton-hopping-like motion determined by DES (tau(DES)) at temperatures around glass transition temperature. The timescales of molecular motions reflected in the tau(c) and tau(DES) were significantly smaller than that of motions leading to molecular rearrangement (molecular rearrangement motions), which correspond to alpha-relaxation. However, the shapes of temperature dependence for the tau(c) and tau(DES) were similar to that of the calorimetrically determined relaxation time of molecular rearrangement motions. Results suggest that the molecular motions reflected in the tau(c) and tau(DES) are linked to molecular rearrangement motions, such that enhancement of molecular rearrangement motions enhances the molecular motions reflected in the tau(c) and tau(DES). Thus, the tau(DES) and tau(c) can reflect changes in molecular mobility leading to unwanted changes in amorphous formulations, and are thought to be a useful measure for evaluating the stability of formulations.     

Characterisation of the glass transition of HPMC using modulated temperature differential scanning calorimetry

The glass transitional behaviour of HPMC powder and film samples has been studied using modulated temperature differential scanning calorimetry (MTDSC) in order to explore the ability of the technique to detect transitions which involve small changes in heat capacity. HPMC E4M Prem samples were studied in both powder and film form using a TA Instruments MDSC 2920 using a range of pans, modulation amplitudes and underlying heating rates. Moisture contents were measured using a TA Instruments TGA 2950. Studies on HPMC powder demonstrated the greater clarity with which the glass transition, seen at approximately 162 degrees C, may be seen using MTDSC compared to conventional DSC. The practical difficulties associated with casting suitable HPMC films are discussed, with similar results for Tg being found for hermetically sealed pans and pin-holed pans. Increasing the modulation amplitude from 0.212 to 0.5 degrees C improved the signal to noise ratio and increased the magnitude of the measured Tg. Increasing the underlying heating rate from 2 to 5 degrees C/min also improved the signal. The study has outlined several features which need to be considered in association with the measurement of HPMC glass transitions using MTDSC; these include the method of sample preparation, the choice of pans, the modulation amplitude and the underlying heating rate.     

Calorimetric investigation of the structural relaxation of amorphous materials: evaluating validity of the methodologies

Although the potential advantages of the amorphous solid state is widely recognized among pharmaceutical researchers, its industrial applications have been mainly limited to freeze-dried injectable formulations where the amorphous form is naturally produced. Applications in oral dosage forms have been limited due, at least in part, to the poor state of knowledge regarding physical properties and stability of amorphous materials. Relaxation behavior is perhaps one of the most important physical characteristics of amorphous materials because relaxation kinetics are closely related to physical and chemical stability. Although recent developments in calorimetry methodology have facilitated detailed characterization of relaxation behavior, some experimental difficulties remain, and quantitative analysis of structural relaxation is still under development. This review focuses on the calorimetric investigation of the structural relaxation of drugs and excipients, and discusses the difficulties in the experimental evaluation of the relaxation time by those methods. We also present an original investigation of the impact of increases in relaxation time during an annealing experiment on the values of relaxation time, tau, and stretched exponential constant, beta, obtained from analysis of the experiment according to the Kohlraush-Williams-Watts kinetic model. Using results from a numerical simulation, we find that the values of tau and beta obtained from the data analysis are too large and too small, respectively, but the value of stretched relaxation time, tau(beta), remains reliable. The time dependence of the relaxation time is likely to play an important role in the non-Arrhenius behavior of pharmaceutical glasses.     

The assessment of the relaxation behaviour of frozen aqueous solutions of human serum albumin and polyvinylpyrrolidine.

In this investigation, the structure and behaviour of frozen solutions of human serum albumin (HSA) alone and in combination with the cryoprotectant polyvinylpyrrolidone (PVP) have been studied using low frequency dielectric analysis and modulated temperature differential scanning calorimetry (MTDSC). Solutions of HSA (1-10 w/v%) and combined solutions (from 1 to 5 w/v% of each ingredient) were prepared and studied thermally and dielectrically over a frequency range of 10(5)-10(-2)Hz every 10 degrees C from +20 to -70 degrees C. Dielectric data were fitted according to the Dissado-Hill theory and the relaxation times calculated. In addition, a relaxation peak was noted for the frozen HSA systems at a frequency approximately one order of magnitude lower than that seen for the PVP systems, with the PVP dominating the response of the mixed systems. The systems showed Arrhenius behaviour, with, for example, the 5% HSA solution showing an activation energy for the relaxation process of 19.34 kJ/mol. In accordance with previous studies on frozen aqueous solutions of PVP, the results suggest that unfrozen water dominated the dielectric response, with the local environment surrounding the HSA being strongly influenced by the PVP. MTDSC data indicated that the PVP and HSA interact in a complex manner in solution, with a glass transition attributable to PVP being seen only in those systems where PVP was present in sufficient excess. In conclusion, the study has suggested that MTDSC and dielectric spectroscopy may be the useful complementary tools with which the structure and molecular mobility of frozen proteinaceous systems may be studied.     

Molecular Mobility of Amorphous Pharmaceutical Solids Below Their Glass Transition Temperatures

Purpose. To measure the molecular mobility of amorphous pharmaceutical solids below their glass transition temperatures (Tg), using indomethacin, poly (vinyl pyrrolidone) (PVP) and sucrose as model compounds. Methods. Differential scanning calorimetry (DSC) was used to measure enthalpic relaxation of the amorphous samples after storage at temperatures 16-47 K below Tg for various time periods. The measured enthalpy changes were used to calculate molecular relaxation time parameters. Analogous changes in specimen dimensions were measured for PVP films using thermomechanical analysis. Results. For all the model materials it was necessary to cool to at least 50 K below the experimental Tg before the molecular motions detected by DSC could be considered to be negligible over the lifetime of a typical pharmaceutical product. In each case the temperature dependence of the molecular motions below Tg was less than that typically reported above Tg and was rapidly changing. Conclusions. In the temperature range studied the model amorphous solids were in a transition zone between regions of very high molecular mobility above Tg and very low molecular mobility much further below Tg. In general glassy pharmaceutical solids should be expected to experience significant molecular mobility at temperatures up to fifty degrees below their glass transition temperature.     

Stability prediction of amorphous benzodiazepines by calculation of the mean relaxation time constant using the Williams-Watts decay function.

The enthalpic relaxation of three amorphous benzodiazepines, diazepam, temazepam and triazolam was studied using differential scanning calorimetry for ageing temperatures which were below the glass transition temperature, and ageing times up to 16 h. Experimental determination of the relaxation enthalpy and the heat capacity change, both accompanying the glass transition, enabled us to calculate the extent of relaxation of the amorphous drugs at specific ageing conditions. Fitting of the relaxation function to the Williams-Watts two parameter decay function led to calculation of the mean relaxation time constant tau and the molecular relaxation time distribution parameter beta. The mean relaxation time constants for the three drugs increased from approximately ten h at the glass transition temperature with more than eight orders of magnitude at 66 K below the glass transition temperature. It was found that the benzodiazepines exhibited significant molecular mobility until approximately 50 K below the glass transition temperature; below this temperature molecular mobility becomes unimportant with respect to the shelf life stability. Hence the presented procedure provides the formulation scientist with a tool to set storage conditions for amorphous drugs and glassy pharmaceutical products.     

Different measures of molecular mobility: comparison between calorimetric and thermally stimulated current relaxation times below Tg and correlation with dielectric relaxation times above Tg

Stability of the amorphous state has been linked to molecular mobility of the matrix; however different techniques may capture different mobility substates. Our previous work suggested that two calorimetric techniques, Isothermal Microcalorimetry (TAM) and MDSC, measured different aspects of mobility with TAM measuring, in part, some faster modes of relaxation in addition to the modes mobilized at T(g). The aim of this work is to compare the relaxation times obtained using Thermally Stimulated Depolarization Current Spectroscopy (TSDC) with calorimetric mobility measured below T(g) and to determine if all measures of relaxation times below T(g) are consistent with relaxation times obtained above T(g) using Dielectric Spectroscopy (DRS). Model compounds were indomethacin, ketoconazole, nifedipine, flopropione, felodipine. For all compounds, relaxation times obtained using Thermal Windowing-TSDC technique below T(g) correlated well with relaxation times (tau) obtained above T(g) by DRS. At any given temperature below T(g), relaxation times measured depended upon the technique used and were in the following order TSDC < TAM < MDSC (tau). TSDC captures some faster relaxations not measured by calorimetric techniques, and therefore, different techniques give different measures of relaxation times below T(g). This information is important in understanding the relationships between mobility in the glassy solid and pharmaceutical stability.     

Characterization of ciclosporin A loaded poly (D,L lactide-co-glycolide) microspheres using modulated temperature differential scanning calorimetry

The aim of this study was to investigate the physical structure of poly (D,L lactide-co-glycolide) (PLGA) microspheres loaded with ciclosporin A in terms of the amorphous properties of the individual components and the phase separation characteristics of the binary systems. Microspheres were prepared using a standard oil-in-water emulsion technique. The thermal properties of the PLGA, ciclosporin A and loaded spheres were investigated using modulated temperature differential scanning calorimetry (MTDSC) using a TA Instruments MTDSC 2920, with scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and high-performance liquid chromatography used as supportive techniques. MTDSC indicated a glass transition for ciclosporin A in the reversing heat flow signal at 107 degrees C, supported by temperature cycling studies, while XRD showed clear evidence for diffraction peaks, thereby indicating that the material as received is semi-crystalline. The unloaded PLGA spheres showed a glass transition (Tg) at 43 degrees C, with no reduction in Tg being observed on loading the peptide up to 50%, w/w. Similarly, no evidence for diffraction peaks were seen for the drug-loaded systems, although the glass transition corresponding to the peptide was observed for the loaded microspheres, suggesting that the drug is present as a separate amorphous phase. Similarly, SEM studies showed the appearance of distinct "islands" on the surface of the spheres that are suggested to correspond to the drug phase, with the size of the islands increasing with drug loading. Evidence is therefore presented that ciclosporin A may exist in a range of solid states, with the degree of crystallinity being altered by processing. In addition, there appears to be little or no miscibility between the drug and PLGA using the manufacturing protocol employed here. These findings may have implications for the choice of manufacturing protocol, the release of peptide drugs from PLGA microspheres and the chemical and physical stability of such drugs.     

Characterization of amorphous ketoconazole using modulated temperature differential scanning calorimetry

The objective of the present study was to characterize the glassy state of ketoconazole and to calculate its molecular mobility below the glass transition, with a view to further developing the use of modulated temperature differential scanning calorimetry (MTDSC) as a means of studying relaxation behavior. Particular emphasis is placed on identifying the influence of the choice of experimental parameters on the measured values of both the glass transition temperature (T(g)) and the relaxation enthalpy magnitude. Amorphous ketoconazole was studied using an amplitude of +/-0.212 K, a period of 40 s, and an underlying heating rate of 2 K/min. The correction required for the calculation of the relaxation endotherm magnitude (the "T(g) shift effect") was demonstrated and is discussed in terms of the mechanism underpinning this phenomenon. Similarly, the influence of the choice of MTDSC experimental parameters on the measured T(g) was studied by varying the amplitude from +/-0.011 to +/-0.424 K and the period from 25 to 50 s. The influence of the cooling rate from the melt on the magnitude of the relaxation endotherm and position of the glass transition was investigated. It was noted that the magnitude of the relaxation endotherm increased with slower cooling rates, this being ascribed to a combination of annealing during the cooling and heating cycle and a further facet of the T(g) shift effect. Annealing experiments were performed at aging temperatures T(g)-12--T(g)-42 K for periods ranging from 10 min up to 16 h. The relaxation behavior was characterized by fitting the calculated extent of relaxation to the Williams-Watts equation. Overall, the study has highlighted theoretical and experimental issues that need to be considered when using both DSC and MTDSC for the calculation of relaxation times.     

Effect of sorbitol and residual moisture on the stability of lyophilized antibodies: Implications for the mechanism of protein stabilization in the solid state

PURPOSE: To investigate the effect of plasticizers on the stability of protein formulations in the solid state and to apply these results to a study of mechanisms of protein stabilization by sugars in the solid sate. METHODS: The IgG1 antibody was formulated with either sucrose or trehalose alone or a mixture of sorbitol with sucrose or trehalose. After lyophilization, the pure protein and sucrose formulations were equilibrated at different relative humidities giving residual moistures from less than 1% to 5% for sucrose systems and up to 17% for pure protein systems. All the samples were stored at 50 degrees C for up to1 month and at 40 and 25 degrees C for up to 6 months. Aggregation and chemical degradation were monitored by size exclusion chromatography (SEC) and ion exchange chromatography (IEX), respectively. The secondary structure was characterized by FTIR using second derivative analysis of Amide I region. Structural relaxation times, tau, an indication of molecular mobility in the glassy matrix, were characterized using the thermal activity monitor (TAM). The tau values of the recombinant human monoclonal antibody (rhuMab) formulation with various water contents were also measured in this study and compared with stability data taken from the literature (Breen ED, Curley JG, Overcashier DE, Hsu CC, Shire SJ, 2001, Pharm Res 18:1345-1353). RESULTS: The structural relaxation time, tau, decreased sharply with increasing water content. However, the stability data suggest a minimum in degradation rate at 2%-3% water content. Addition of a small amount of sorbitol to a sucrose-based formulation resulted in greater retention of native structure, smaller relaxation time, but improved stability. However, with the trehalose-based formulations, addition of sorbitol had no effect on protein structure (FTIR), but the decrease in relaxation time and the improvement in stability were qualitatively similar to the corresponding data obtained with the sucrose-based formulation. CONCLUSION: Glass dynamics as measured by tau could not explain the stability results. Stability correlated best with the preservation of native structure for sucrose-based formulations, but with the trehalose-based formulation, neither structural relaxation time nor extent of native structure was predictive of stability. However, it is possible that the beta-relaxations rather than the alpha-relaxation (i.e., the tau we measured) is critical to the stability. Plasticizers like glycerol may decrease tau for "alpha-motion" but increase tau for "beta-motion" and stabilize proteins (Cicerone MT, Tellington A, Trost L, Sokolov A, 2003, BioProcess Inter 1:1-9).     

Interpretation of relaxation time constants for amorphous pharmaceutical systems

The molecular mobility of amorphous pharmaceutical materials is known to be a key factor in determining their stability, reactivity, and physicochemical properties. Usually such molecular mobility is quantified using relaxation time constants. Typically relaxation processes in amorphous systems are non-exponential and relaxation time constants are usually obtained from experimental data using a curve fitting procedure involving the empirical Kohlrausch-Williams-Watts (KWW) equation. In this article we explore the possible relationship between the KWW curve fitting parameters (tau(KWW), beta(KWW)) and common statistical measures of the average and the distribution (e.g., median, standard deviation) of the relaxation time values. This analysis is performed for several common statistical distributions (e.g., normal, lognormal, and Lorentzian), and the results are compared and analyzed in the context of pharmaceutical product stability predictions. The KWW function is able to describe relaxation processes stemming from several different statistical distribution functions. Under some circumstances the "average" relaxation time constant of the KWW equation (tau(KWW)) is significantly different from common statistical measures of the central value of a distribution (e.g., median). Simply knowing the relaxation time constants from the fit of the KWW equation is not sufficient to completely characterize and quantify the molecular mobility of amorphous pharmaceutical materials. An appreciation of the distribution of relaxation times and the resulting effects upon the KWW constants should be considered to be essential when working with amorphous pharmaceutical materials, especially when attempting to use relaxation time constants for predicting their physical or chemical stability.