Glass fragility and the stability of pharmaceutical preparations--excipient selection.

The objectives of this study were, first, to calculate the zero mobility temperatures, T0, of trehalose and sucrose by the Pikal method from the width of the glass transition and compare these to the literature, obtained by enthalpy relaxation measurement, and second, to compare the T0 values and physicochemical properties of trehalose to those of sucrose in terms of potential to stabilize labile actives in the glassy state. Differential scanning calorimetry and coulometric Karl-Fischer analysis were used. The glass transition temperatures, Tg, for the two carbohydrates at circa 0.7% moisture were 101 degrees C and 64 degrees C for trehalose and sucrose, respectively. Anhydrous amorphous trehalose had a Tg of 116 degrees C. The T0 values were found to be 44 and 3.5 degrees C for trehalose and sucrose, respectively. The Tg-T0 value for sucrose was compared, and found to be in good agreement with that found by enthalpy relaxation measurements. Trehalose was found to be resistant to crystallization above the glass temperature. The study supports the validity of the calculation method proposed by Pikal for T0. It has been proposed in the literature that T0 is a better measure of stability than Tg. Trehalose has a significantly higher T0 than sucrose and thus would work more effectively in stabilizing a labile active.

Determination of the unfrozen water content of maximally freeze-concentrated carbohydrate solutions.

The heat capacity change at T'g has been studied in freeze-concentrated carbohydrate solutions. The values obtained have been compared with those found for high concentration solutions that do not undergo freezing above Tg. The analysis has indicated that the freezing process influences the degree of stress in the glassy phase. This results in a complex power-time curve when frozen solutions are heated in a differential scanning calorimeter. The endotherm produced by the stress relaxation can cause considerable error in W'g measurement obtained by any method that relies on the integration of the power-time curve. A more reliable method for W'g determination is via the intersection of T'g with a previously prepared Tg/Wg calibration curve.

Cold destabilisation of enzymes.

The thermal stability profile of chymotrypsinogen has been investigated in the temperature range 230-340 K, with special emphasis on the phenomenon of cold instability. Differential scanning calorimetry was used to study the heat capacities of the native and denatured protein in undercooled solution and the results were combined with those obtained by spectrophotometry at ordinary temperatures. The partial heat capacities of both forms decrease with decreasing temperature, assuming negative values. In the experimentally accessible temperature range (above the spontaneous nucleation temperature of ice) the heat capacity difference delta C is found to be positive with a non-linear temperature dependence. delta C is predicted to change sign at some low temperature which cannot, however, be reached by experiment for chymotrypsinogen. In contrast to earlier studies, covering a much more limited temperature range and having to employ an additional destabilisation by means of pH and/or chaotropes, the present findings permit the construction of a more reliable thermodynamic stability profile and related properties. These differ in important details from those reported for other proteins, but based on measurements only in the neighbourhood of the heat-denaturation temperature. The thermodynamic characteristics are, however, in good agreement with earlier predictions and with recent low-temperature measurements on the tetrameric enzyme lactate dehydrogenase.

The effective use of differential scanning calorimetry in the optimisation of freeze-drying processes and formulations.

The formulation and processing variables required for the effective freeze-drying of labile biologicals can be determined from an understanding of the physics of freeze-concentration. Three measurable values, Tg' (the glass temperature associated with maximum freeze-concentration), Wg' (the unfrozen water at this temperature) and Tg (the glass temperature of the dried product) are the keystones. These can be obtained by differential scanning calorimetry. This paper describes their measurement. Techniques to increase the detectability of glass transitions are discussed. Method of calculating Wg' from DSC power-time curves are critically reviewed. An example is presented in which it is demonstrated how failure to perform primary drying below the glass transition results in products on inferior quality. Finally, it is described how measurements of the glass temperature of the "dry" product can be used to predict storage stability. The limitation of using moisture determination to predict stability is high-lighted.

Water, temperature and life.

Cold is the fiercest and most widespread enemy of life on earth. Natural cold adaptation and survival are discussed in terms of physicochemical and biochemical water management mechanisms, relying on thermodynamic or kinetic stabilization. Distinctions are drawn between general effects of low temperature (chill) and specific effects of freezing. Freeze tolerance is a misnomer because tolerance does not extend to the cell fluids. Freezing is confined to the extracellular spaces where it acts as a means of protecting the cytoplasm against freezing injury. Freeze resistance depends on the phenomenon of undercooling, a survival mechanism that relies on the long-term maintenance of a thermodynamically highly unstable state. Correct water management involves many factors, among them the control of membrane composition and transmembrane osmotic equilibrium, the biosynthesis of compounds able to afford protection against injury through freeze desiccation and the availability (or inactivation) of biogenic ice nucleation catalysts.

The cold-induced denaturation of lactate dehydrogenase at sub-zero temperatures in the absence of perturbants.

The cold-induced denaturation of lactate dehydrogenase has been determined in an unfrozen, cryoprotectant free solution at sub-zero temperatures. The cold-induced denaturation temperature (TL) has been found to be -28 degrees C. The results for the first time clearly establish that temperature alone can induce denaturation in a cooled protein solution. The validity of earlier data, obtained in the presence of perturbants (particularly pH or guanidinium chloride), is discussed.

The effect of aqueous methanol cryosolvents on the heat- and cold-induced denaturation of lactate dehydrogenase.

The effect on the low-temperature-induced denaturation temperature of various concentrations of methanol has been studied for lactate dehydrogenase. The results have been compared to similar data for the thermal denaturation temperature. Extrapolations of the low-temperature data show that, in a physiological buffer in the absence of methanol, the cold denaturation temperature would be -30 degrees C. Data obtained with high concentrations of methanol indicate that residues are exposed to a similar degree upon either heat- or cold-induced denaturation. Aggregation does not occur in the cold-denatured protein. Cold-induced denaturation is fully reversible at a protein concentration of 250 micrograms/ml. The spectra of the two denatured forms are similar.

Variation in apparent enzyme activity in two-enzyme assay systems: phosphoenolpyruvate carboxylase and malate dehydrogenase.

We have employed the two-enzyme assay system for phosphoenolpyruvate to investigate the effect on the apparent phosphoenolpyruvate carboxylase (PEP-C) activity of the use of malate dehyrogenase (MDH) that has been stabilized in either glycerol or (NH4)2SO4. The type of MDH stabilizer has a marked effect on the apparent activity of the PEP-C. The apparent activities of the PEP-C are 1.34 and 0.43 U/mg in the presence of glycerol and salt-stabilized MDH, respectively. The implications of the observations for diagnostic assays are discussed.

The thermodynamics of protein stability. Cold destabilization as a general phenomenon.

A theoretical analysis of the temperature/stability profiles of proteins shows that, where a two-state model represents the denaturation, and where the free energy of denaturation delta G(T) shows a strong temperature dependence, then the protein becomes subject to both high- and low-temperature destabilization. In the simplest case delta G(T) is parabolic, therefore the high temperature TH, where delta (G(TH) = 0, is complemented by a low temperature TL, where delta G(TL) = 0. It is generally stated that the partial molal heat capacity change delta C accompanying the heat denaturation is positive and independent of the temperature. This implies that heating the protein through TL results in a negative delta C which seems physically unsatisfactory. The constant delta C model is explored and a physically more realistic model is advanced which allows for a temperature-dependent delta C which changes sign at some temperature within the range of stability of the native protein; delta G(T) then has the form of a skewed parabola. Experimental heat capacity data for native lysozyme and for a flexible polymer lend support to this model. The molecular basis of cold inactivation of proteins is discussed in the light of the thermodynamic analysis.

Subzero-temperature preservation of reactive fluids in the undercooled state. II. The effect on the oxidation of ascorbic acid of freeze concentration and undercooling.

The rate of oxidation of ascorbic acid has been measured in both frozen and undercooled solutions. A new interpretation is advanced for changes in the rate of ascorbic acid oxidation in freeze-concentrated solutions. The results obtained with undercooled solutions indicate a rate reduction in line with that predicted by the Arrhenius equation. It is also demonstrated that undercoohng provides a method for greatly extending the shelf life of reactive fluids.

Subzero-temperature preservation of reactive fluids in the undercooled state. I. The reduction of potassium ferricyanide by potassium cyanide.

Many reactions show enhanced rates at subzero temperatures due to freeze concentration. The reduction of potassium ferricyanide by potassium cyanide has been studied at subzero temperatures in both the undercooled and the frozen state. The pseudo-first-order rate constants calculated differ greatly from those in previous reports. A high degree of freeze concentration and supersaturation in frozen bulk solutions occurs. It has been clearly demonstrated that undercooled preservation provides a useful method for the long-term storage of reactive mixtures.

Preservation of viable cells in the undercooled state.

Previous studies into the mechanisms governing the freezing of cells in the absence of extracellular ice have been extended to develop a method for the preservation of viable cells in the undercooled state. Deep undercooling of cells is achieved by suspending fine droplets of the cells in oil to make an emulsion, thus minimizing initiation of extracellular ice nucleation. Attempts to preserve yeast cells, cultured sainfoin cells, and dissected shoot-tips (pea and potato) in this way are described. The main findings are that yeast cells can be preserved undercooled at -20 degrees C for at least 16 weeks with no detectable loss of viability, showing that -20 degrees C is a low enough temperature for inhibition of significant biochemical deterioration and that the emulsions are stable over long periods. In preliminary experiments, sainfoin cells survived 24 hr at -10 degrees C, and shoot-tips survived 48 hr at -10 degrees C. Sainfoin cells, conditioned by growth in medium supplemented with sorbitol, showed enhanced survival after exposure to low temperatures and a lower intracellular freezing point than control cells. Possible reasons for this are discussed.