Studies of the Crystallization of Amorphous Trehalose Using Simultaneous Gravimetric Vapor Sorption/Near IR (GVS/NIR) and “Modulated” GVS/NIR

The purpose of this research was to investigate the influence of changes in the amorphous state on the crystallization of trehalose. Amorphous trehalose is known to stabilize biomaterials; hence, an understanding of crystallization is vital. Amorphous trehalose, prepared by spray-drying, was exposed to either a single step (0–75%) in relative humidity (RH) or to modulated 0–75–0% RH to cause crystallization. For the single-step experiment, two samples crystallized in a predictable manner to form the dihydrate. One sample, while notionally identical, did not crystallize in the same way and showed a mass loss throughout the time at 75% RH, with a final mass less than that expected for the dihydrate. The idiosyncratic sample was seen to have a starting near infrared (NIR) spectra similar to that exhibited by anhydrous crystalline trehalose, implying that short-range order in the amorphous material (or a small amount of crystalline seed, not detectable using powder X-ray diffraction) caused the sample to fail to form the dihydrate fully when exposed to high RH. The modulated RH study showed that the amorphous material interacted strongly with water; the intensity of the NIR traces was not proportional to mass of water but rather the extent of hydrogen bonding. Subsequent crystallization of this sample clearly was a partial formation of the dihydrate, but with the bulk of the sample then shielded such that it was unable to show significant sorption when exposed to elevated RH. It has been shown that the nature of the amorphous form will alter the way in which samples crystallize. With oscillation in RH, it was possible to further understand the interactions between water and amorphous trehalose.     

Modification of the Solid-State Nature of Sulfathiazole and Sulfathiazole Sodium by Spray Drying

Solid-state characterisation of a drug following pharmaceutical processing and upon storage is fundamental to successful dosage form development. The aim of the study was to investigate the effects of using different solvents, feed concentrations and spray drier configuration on the solid-state nature of the highly polymorphic model drug, sulfathiazole (ST) and its sodium salt (STNa). The drugs were spray-dried from ethanol, acetone and mixtures of these organic solvents with water. Additionally, STNa was spray-dried from pure water. The physicochemical properties including the physical stability of the spray-dried powders were compared to the unprocessed materials. Spray drying of ST from either acetonic or ethanolic solutions with the spray drier operating in a closed cycle mode yielded crystalline powders. In contrast, the powders obtained from ethanolic solutions with the spray drier operating in an open cycle mode were amorphous. Amorphous ST crystallised to pure form I at ≤35 % relative humidity (RH) or to polymorphic mixtures at higher RH values. The usual crystal habit of form I is needle-like, but spherical particles of this polymorph were generated by spray drying. STNa solutions resulted in an amorphous material upon processing, regardless of the solvent and the spray drier configuration employed. Moisture induced crystallisation of amorphous STNa to a sesquihydrate, whilst crystallisation upon heating gave rise to a new anhydrous polymorph. This study indicated that control of processing and storage parameters can be exploited to produce drugs with a specific/desired solid-state nature.KEY WORDS: amorphous state, dynamic vapour sorption, particle habit, physical stability, polymorphism, sulfathiazole     

An examination of the moisture sorption characteristics of commercial magnesium stearate

The objective of this study was to characterize the moisture sorption of magnesium stearate and the morphological changes, if any, resulting from moisture sorption. Six samples of commercial magnesium stearate USP were examined. Moisture sorption isotherms were obtained at 25°C and 5% to 98% relative humidity (RH) using a moisture balance. Changes in crystal form resulting from moisture sorption were determined by x-ray diffraction. There were differences in the shape of the isotherm, reversibility of moisture uptake, and shape of the hysteresis loop in the isotherms of crystalline and amorphous magnesium stearates. The isotherm of crystalline magnesium stearate was almost parallel to the pressure axis until and RH of ∼80%. The isotherm of the amorphous sample was characterized by continuous uptake of water over the entire range of RH. Exposure of amorphous magnesium stearate to RH greater than 70% resulted in the formation of the trihydrate. The trihydrate was converted into the anhydrous form when heated to a temperature of 100°C to 105°C. The trihydrate could be generated by exposing the anhydrate to RH higher than 70%.     

Aufnahme von Wasserdampf aus ungesättigter Atmosphäre und ihre ökologische Bedeutung bei mesostigmaten Milben

A number of mesostigmatic mites of diverse biologies and habitats absorb water vapour from unsaturated atmospheres. This includes haematophagous, predatory, saprophagous and polyphagous species. The lowest humidity at which vapour uptake is possible ranges from 76 to 97% r.h. depending on the species. The transpiration rates differ by a factor of 50 among different species. By means of sorption and transpiration the water content of the mites follows variations in the ambient humidity rather immediately. Uptake of atmospheric water compensates water losses of the mites. The critical equilibrium humidity of the mites marks the relative humidity of the ambient air at which sorption and transpiration counterbalance each other and at which the mites are capable of maintaining body water equilibrium. An oral site of vapour sorption is indicated. Sorption and transpiration are physiological parameters of ecological relevance related to increase of populations, survival and habitat preferences of these mites significant as parasites and biological control agents.     

Water uptake in the cat flea Ctenocephalides felis (Pulicidae: Siphonaptera)

To counteract water loss due to excretion, cuticular transpiration and respiration, various groups of arthropods have developed mechanisms for active uptake of water vapor from unsaturated air. In this study, active uptake capabilities and water loss rates were examined in the various developmental stages of the cat flea, Ctenocephalides felis. To determine critical equilibrium humidity, the lowest relative humidity at which active water uptake can occur, pre-desiccated immature and adult fleas were placed in a series of humidity regimes ranging from 44 to 93% RH. Active uptake occurred in larval stages at relative humidities above 53% and in pre-pupae at 75-93% RH. Pupae and adults did not demonstrate active uptake at any humidity. Optimal uptake for larvae occurred between 20 and 30 degrees C. When placed over Drierite (<10% RH), larval and adult stages demonstrated a higher rate of water loss than pre-pupal and pupal stages. Active water uptake is necessary to ensure proper development of the larvae of C. felis. Active uptake ceases after the larval-pupal ecdysis and it appears that adults have lost the ability to actively uptake water.     

A comparison of the water balance characteristics of Typhlodromus occidentalis and Amblyseius finlandicus mites (Acari: Phytoseiidae) and evidence for the site of water vapour uptake

To determine how adult Typhlodromus occidentalis, a mite used in the biological control of spider mites, thrive in arid western North America, the water balance characteristics of adult females were compared to those of a laboratory colony of Amblyseius finlandicus originating from Finnish apple orchards. The mites contained comparable amounts of water (73.6 and 74.9%, respectively, for T. occidentalis and A. finlandicus), absorbed water from the air between 86% and 92% relative humidity (RH) (critical equilibrium humidity) and drank free water from droplets. Typhlodromus occidentalis were distinguished, however, by having lower net water loss rates (0.8% versus 1.3% h–1 at 0% RH, 20°C for A. finlandicus), a feature that enables them to retain water more effectively. Above the critical equilibrium humidity water was lost by adult female T. occidentalis whose mouth parts had been blocked with wax, implying an oral uptake mechanism for the absorption of water vapour. © Rapid Science Ltd. 1998     

Moisture Sorption Isotherms of Various Tobaccos

The equilibrium moisture contents of sun-cured (Kroumougrad), flue-cured (Bright Yellow—4) and air-cured (Burley-21 and Matsukawa) tobaccos were measured over a relative humidity range from 5 to 80% at 20°C. The moisture sorption isotherms of tobaccos were of sigmoid type, and classified into two groups. In a lower humidity range below ca. 40% RH, the A group (Kroumougrad and BY-4) had a smaller moisture sorption capacity than B group (Burley-21 and Matsukawa), while in a higher humidity range above ca. 50% RH the former had a larger moisture sorption capacity than the latter. By extracting with water, the moisture content of BY-4 was increased in the lower humidity range, while it decreased in the higher humidity range. However, the moisture content of Matsukawa was scarecely changed by extracting it with water. These results suggest that the differences in equilibrium moisture content with the type of curing were due to the differences in contents of water soluble com- ponents. To control the hygroscopic properties of a tobacco, therefore, the influences of the addition of sucrose and glycerol on the equilibrium moisture content were quantitatively analysed. The moisture sorption capacity of tobacco was greatly different from its nitrogen sorption capacity. The specific surface area of tobacco calculated from moisture sorption isotherm was ca. 110 times larger than the specific surface area calculated from the nitrogen sorption isotherm. Both the nitrogen and moisture sorption data should be necessary for better understanding of the complicated sorption-desorption phenomena in tobaccos.     

A digital registration system for net photosynthesis and transpiration measurements in the field and an associated analysis of errors

Summary A digital registration system used with temperature- and humidity-controlled cuvettes for net photosynthesis and transpiration measurements in the field is described. The associated errors of the measured parameters and calculated data are estimated. The digitalization is based on an analogue registration which is of primary importance in the control of experimental conditions in the cuvettes. The digital system is connected to the analogue registration in series. The error associated with digitalization is 0.1% across 70% of the scale. This error increases to 0.2% between 3 and 30% on the scale due to a minor lack of linearity. The reproducibility of the digitalization is ±0.024%.The error associated with data transfer in the digitalization and the errors of the analogue registration are estimated for temperature and humidity measurements (error of air and leaf temperature is ±0.1° C; error of the dew point temperature is ±1.1° C dew point). The effect of these errors on the calculation of relative humidity and the water vapour difference between the leaf and the air is determined using the progressive error law. At 30° C and 50% relative humidity, the error in relative humidity is ±7.4%, the error for the water vapour difference is ±6.6%. The dependence of these errors on temperature and humidity is shown.The instrument error of the net photosynthesis measurement is calculated to be ±4.2%. Transpiration measurements have an average inaccuracy of ±8.3%. The total diffusion resistance which is calculated from values of transpiration and the water vapour difference has an average error of ±10.9%. The sizeable influence of errors in humidity and temperature measurements on the calculated diffusion resistance is demonstrated. The additional influence of biological errors associated with field measurements is discussed.     

Physicochemical Characterization and Water Vapor Sorption of Organic Solution Advanced Spray-Dried Inhalable Trehalose Microparticles and Nanoparticles for Targeted Dry Powder Pulmonary Inhalation Delivery

Novel advanced spray-dried inhalable trehalose microparticulate/nanoparticulate powders with low water content were successfully produced by organic solution advanced spray drying from dilute solution under various spray-drying conditions. Laser diffraction was used to determine the volumetric particle size and size distribution. Particle morphology and surface morphology was imaged and examined by scanning electron microscopy. Hot-stage microscopy was used to visualize the presence/absence of birefringency before and following particle engineering design pharmaceutical processing, as well as phase transition behavior upon heating. Water content in the solid state was quantified by Karl Fisher (KF) coulometric titration. Solid-state phase transitions and degree of molecular order were examined by differential scanning calorimetry (DSC) and powder X-ray diffraction, respectively. Scanning electron microscopy showed a correlation between particle morphology, surface morphology, and spray drying pump rate. All advanced spray-dried microparticulate/nanoparticulate trehalose powders were in the respirable size range and exhibited a unimodal distribution. All spray-dried powders had very low water content, as quantified by KF. The absence of crystallinity in spray-dried particles was reflected in the powder X-ray diffractograms and confirmed by thermal analysis. DSC thermal analysis indicated that the novel advanced spray-dried inhalable trehalose microparticles and nanoparticles exhibited a clear glass transition (T _g). This is consistent with the formation of the amorphous glassy state. Spray-dried amorphous glassy trehalose inhalable microparticles and nanoparticles exhibited vapor-induced (lyotropic) phase transitions with varying levels of relative humidity as measured by gravimetric vapor sorption at 25°C and 37°C.     

Occurrence,properties and biological implications of the active uptake of water vapour from the atmosphere in Psocoptera

Water vapour absorption is shown to occur in 22 species of Psocoptera inhabiting diverse environments and representing all major groups of this insect order. Evidently the faculty is a common feature of the whole order and it seems not to be related to specific environmental conditions. For the first time water vapour uptake could be demonstrated in fully winged and flying insects. The critical equilibrium humidities vary considerably among different species ranging from 58 to 85% r.h. Marked interspecific differences are also observed in water loss and uptake rates but no clear correlation with habitat or systematic group is recognizable. The uptake rates of Psocoptera are among the highest of all arthropods investigated so far. From weight recordings with a sensitive microbalance it could be seen that continuous operation of the uptake mechanism is restricted to limited periods of time of less than 1 hr regardless of the water status of the animals. Initiation and termination of the uptake process are abrupt and continuous uptake proceeds at a constant rate at a given relative humidity. Uptake rates are humidity-dependent decreasing with falling relative humidity whereas the adjustment of the equilibrium level of body water is independent of ambient humidity. Equilibrium is maintained by intermittent operation of the uptake mechanism within ca. 3% of body water mass. The uptake mechanism exhibits marked sensitivity to starvation in most members of the Psocomorpha. Some features of the uptake process of Psocoptera are in close agreement with those of Mallophaga reflecting the close relationship between the two groups.     

Monitoring Phase Transformations in Intact Tablets of Trehalose by FT-Raman Spectroscopy

The purpose of this study is to monitor phase transformations in intact trehalose tablets using FT-Raman spectroscopy. Tablets of trehalose dihydrate, amorphous trehalose (obtained by freeze-drying aqueous trehalose solutions), and anhydrous trehalose (β-trehalose) were prepared. The tablets were exposed to different conditions [11% and 0% RH (60°C); 75% RH (25°C)] and monitored periodically over 96 h using Raman spectroscopy. Within 96 h of storage, the following phase transformations were observed: (1) trehalose dihydrate → β-trehalose (11% RH, 60°C), (2) trehalose dihydrate → α-trehalose (0% RH, 60°C), (3) β-trehalose → trehalose dihydrate (75% RH, 25°C), and (4) amorphous trehalose → trehalose dihydrate (75% RH, 25°C). FT-Raman spectroscopy was a useful technique to identify the solid form and monitor multiple-phase transformations in intact trehalose tablets stored at different conditions.     

Factors influencing changes in moisture content during storage of freeze-dried vaccines in vials

Previous studies have demonstrated that the water content of freeze-dried vaccines increases during storage. The reasons for these variations in water content are discussed in this paper. Different possible mechanisms have been considered: microleakages at the closure sealing point, water vapour transfer through the closure (permeation, loss and uptake of water by the stopper). Several types of vials and closures were studied, under different conditions of storage. Regardless of the vial form selected, the probability of microleakages was low. The stopper would seem to play an important role in the water transfer mechanisms. The moisture content of the stopper determines its equilibrium relative humidity (ERH) and whether there will be moisture transfer between the stopper and the vial contents and between the stopper and the storage atmosphere.     

Quantitative determination of water sorption by plant cuticles

Abstract. The water sorption by cuticular membranes (CM) isolated from both leaves and fruit and representing different structural and chemical cuticular types, was measured over the whole range of relative humidities using a magnetic suspension microbalance. The sorption isotherms were generally not linear and sorption increased more rapidly at the highest values of relative humidity. The highest values of water content, at 80–99% r.h., measured for the seven species ranged from 1.1 to 7.7% of the dehydrated weight. Extraction of the soluble cuticular lipids did not result in a decrease of sorption, but only in a lower interspecific dispersion. There was a drastic reduction (63%) in water sorption when polymer matrix membranes (MX) were partially acid-hydrolysed; but methylation or charging with iron of (MX) did not have any significant effect on water sorption. The data obtained are discussed in relation to cuticular permeability. Two determinants of water permeability were determined: the partition coefficient (K) relating the equilibrium Water concentration of the cuticle to that of the surrounding atmosphere; and the diffusion coefficient (D), calculated from the half-times of the sorption process in kinetic measurements.     

Core-shell encapsulation formulations to stabilize desiccated Bradyrhizobium against high environmental temperature and humidity

Engineered materials to improve the shelf-life of desiccated microbial strains are needed for cost-effective bioaugmentation strategies. High temperatures and humidity of legume-growing regions challenge long-term cell stabilization at the desiccated state. A thermostable xeroprotectant core and hydrophobic water vapour barrier shell encapsulation technique was developed to protect desiccated cells from the environment. A trehalose core matrix increased the stability of desiccated Bradyrhizobium by three orders of magnitude over 20 days at 32°C and 50% relative humidity (RH) compared to buffer alone; however, the improvement was not deemed sufficient for a shelf-stable bioproduct. We tested common additives (skim milk, albumin, gelatin and dextran) to increase the glass transition temperature of the desiccated product to provide further stabilization. Albumin increased the glass transition temperature of the trehalose-based core by 40°C and stabilized desiccated Bradyrhizobium for 4 months during storage at high temperature (32°C) and moderate humidity (50% RH) with only 1 log loss of viability. Although the albumin-trehalose core provided exceptional protection against high temperature, it was ineffective at higher humidity conditions (75%). We therefore incorporated a paraffin shell, which protected desiccated cells against 75% RH providing proof of concept that core and shell encapsulation is an effective strategy to stabilize desiccated cells.     

Fourier transform infared spectroscopy investigation of protein conformation in spray-dried protein/trehalose powders

Spray drying is a way to generate protein solids (powders), which is also true for lyophilization. Sugars are used to protect proteins from conformational changes and chemical degradations arising from drying processes and storage conditions such as the humidity. The influence of trehalose and humidity on the conformation and hydration of spray-dried recombinant human granolucyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon-alpha (rConIFN) was investigated using Fourier transform IR spectroscopy. The spectral analysis of spray-dried powders in the amide I region demonstrated that trehalose stabilized the alpha-helical conformation of both rhG-CSF and rConIFN proteins. Exposure of the pure protein powders to 33% relative humidity (RH) resulted in the formation of beta sheets and loss of turns but no change in alpha-helical structure. Trehalose reduced the magnitude of the changes in beta sheets and turns. Exposure of the pure protein powders to 75% RH resulted in the loss of alpha-helical conformation with a corresponding increase in beta structures (beta sheets and turns). Trehalose did not protect proteins from the loss of alpha-helical structures, but it reduced the formation of antiparallel beta sheets. Hydrogen-deuterium exchange (H-D exchange) was used to further characterize these hydration-induced conformational changes. At 33% RH the percent exchange of the protein decreased with increasing trehalose content, indicating a greater protection of the protein from H-D exchange by a higher concentration of trehalose. Such protection correlates with decreased conformational changes of the protein by trehalose at this humidity. At 75% RH the degree of H-D exchange of the protein was insensitive to the powder composition in all powders. Surprisingly, the H-D exchange of trehalose was low at about 20-25%, which was nearly independent of the protein/trehalose ratio and humidity, indicating that the exchangeable protons on trehalose molecules are highly protected in protein-containing powders. The observed protein hydration is related to the effect of trehalose on the conformational changes of the protein under humidity.     

Solid-vapor interactions: Influence of environmental conditions on the dehydration of carbamazepine dihydrate

The goal of this research was a phenomenological study of the effect of environmental factors on the dehydration behavior of carbamazepine dihydrate. Dehydration experiments were performed in an automated vapor sorption apparatus under a variety of conditions, and weight loss was monitored as a function of time. In addition to lattice water, carbamazepine dihydrate contained a significant amount of physically bound water. Based on the kinetics of water loss, it was possible to differentiate between the removal of physically bound water and the lattice water. The activation energy for the 2 processes was 44 and 88 kJ/mol, respectively. As expected, the dehydration rate of carbamazepine dihydrate decreased with an increase in water vapor pressure. While dehydration at 0% relative humidity (RH) resulted in an amorphous anhydrate, the crystallinity of the anhydrate increased as a function of the RH of dehydration. A method was developed for in situ crystallinity determination of the anhydrate formed. Dehydration in the presence of the ethanol vapor was a 2-step process, and the fraction dehydrated at each step was a function of the ethanol vapor pressure. We hypothesize the formation of an intermediate lower hydrate phase with unknown water stoichiometry. An increase in the ethanol vapor pressure first led to a decrease in the dehydration rate followed by an increase. In summary, the dehydration behavior of carbamazepine dihydrate was evaluated at different vapor pressures of water and ethanol. Using the water sorption apparatus, it was possible to (1) differentiate between the removal of physically bound and lattice water, and (2) develop a method for quantifying, in situ, the crystallinity of the product (anhydrate) phase.     

A new, vapour-phase mechanism for stomatal responses to humidity and temperature

A new mechanism for stomatal responses to humidity and temperature is proposed. Unlike previously-proposed mechanisms, which rely on liquid water transport to create water potential gradients within the leaf, the new mechanism assumes that water transport to the guard cells is primarily through the vapour phase. Under steady-state conditions, guard cells are assumed to be in near-equilibrium with the water vapour in the air near the bottom of the stomatal pore. As the water potential of this air varies with changing air humidity and leaf temperature, the resultant changes in guard cell water potential produce stomatal movements. A simple, closed-form, mathematical model based on this idea is derived. The new model is parameterized for a previously published set of data and is shown to fit the data as well as or better than existing models. The model contains mathematical elements that are consistent with previously-proposed mechanistic models based on liquid flow as well as empirical models based on relative humidity. As such, it provides a mechanistic explanation for the realm of validity for each of these approaches.     

Desiccation tolerance of recalcitrant Theobroma cacao embryonic axes: the optimal drying rate and its physiological basis

Recalcitrant seed axes were reported to survive to lower water contents under fast-drying conditions. The present study was to examine the hypothesis that drying rate and dehydration duration could interact to determine desiccation tolerance through different physico-chemical mechanisms. The effect of drying rate on desiccation tolerance of Theobroma cacao seed axes at 16 degrees C was examined. Rapid-drying at low relative humidity (RH) and slow-drying at high RH were more harmful to cocoa axes, because electrolyte leakage began to increase and axis viability began to decrease at high water contents. Maximum desiccation tolerance was observed with intermediate drying rates at RH between 88% and 91%, indicating the existence of an optimal drying rate or optimal desiccation duration. This maximum level of desiccation tolerance for cocoa axes (corresponding to a critical water potential of -9 MPa) was also detected using the equilibration method, in which axes were dehydrated over a series of salt solutions or glycerol solutions until the equilibrium. These data confirmed that the physiological basis of the optimal drying rate is related to both mechanical stress during desiccation and the length of desiccation duration during which deleterious reactions may occur. The optimal drying rate represents a situation where combined damages from mechanical and metabolic stresses become minimal.     

Characterizing protein structure in amorphous solids using hydrogen/deuterium exchange with mass spectrometry

Elucidating protein structure in amorphous solids is central to the rational design of stable lyophilized protein drugs. Hydrogen/deuterium (H/D) exchange with electrospray ionization mass spectrometry was applied to lyophilized powders containing calmodulin (17 kDa) and exposed to D(2)O vapor at controlled relative humidity (RH) and temperature. H/D exchange was influenced by RH and by the inclusion of calcium chloride and/or trehalose in the solid. The effects were not exhibited uniformly along the protein backbone but occurred in a site-specific manner, with calcium primarily influencing the calcium-binding loops and trehalose primarily influencing the alpha-helices. The results demonstrate that the method can provide quantitative and site-specific structural information on proteins in amorphous solids and on changes in structure induced by protein cofactors and formulation excipients. Such information is not readily available with other techniques used to characterize proteins in the solid state, such as Fourier transform infrared, Raman, and near-infrared spectroscopy.