Study of the effect of the oxytetracycline crystallization conditions on the process indices]

Such factors as the rate of the changes in pH, temperature, mixer speed and the nature of the anions present in the solution has a significant effect on the indices of oxytetracycline dihydrate crystallization, i. e. residual content of the antibiotic in the mother solution and the specific surface of the crystalls. In this connection the effect of the above factors on the main indices of the process were studied. On the basis of the experimental data dependences were found which provided determination of the crystallization conditions securing the process indices.     

Effect of the crystallization conditions of tetracycline base on the properties of the powders and drug forms obtained. The dependence of the degree of dispersion of tetracycline base on the crystallization conditions]

Characteristics of the powder dispersity of tetracycline base samples prepared by directed crystallization with variation of the process conditions were determined by the sedimentation method. It was found that the speed of the solution agitation had the maximum effect on the level and nature of the dispersity. The rate of the solution temperature and pH changing during the crystallization process had also a significant effect at low agitation speed.     

温度影响蛋白质结晶的研究现状

温度控制作为调控蛋白质结晶过程的手段,在结晶实验中被广泛采用。热历史效应作为蛋白质结晶实验中新的影响因素,已被越来越多的科学家所重视。控制温度可以改变蛋白质的溶解度,进一步改变溶液的过饱和度,从而影响结晶过程。我们简要总结了温度对蛋白质结晶的影响及应用温度技术控制蛋白质晶体生长的各种技术,为蛋白质结晶工作提供理论和实验依据。     

The effects of filtration on protein nucleation in different growth media.

Filtration effects of turkey egg white lysozyme solution (TEWL) prior to subjecting it to crystallization conditions are investigated. Filtering TEWL solution and crystallizing it in ungelled media significantly decreased the number of conditions yielding crystals. This decrease dependent on the membrane cut-off used for filtration. From this, the postulated factors aiding in nucleation are estimated to be 0.17 microns in diameter. The existence of these factors was verified by the procedure of reversed filtration: filtered solutions passed through their inverted filter membrane a second time lead to improved crystallization results. The effect of aging of the TEWL solution prior to subjecting it to ungelled crystallization conditions was also verified. We did not find any time-dependent change in the size or the number of crystals per drop. Repeating the filtration experiments in agarose-gelled crystallization media showed that the influence of filtration on the crystallization outcome was significantly diminished. Far better crystallization results were obtained compared to ungelled media. However, there is a certain aging effect linked to filtration in gelled media. Different crystallization results were obtained depending on whether filtration was performed before or after aging and subsequent crystallization. This suggests a secondary time-dependent effect.     

Effect of the permeability of lysosomal and mitochondrial membranes on the ability of their cryoextracts to inhibit the protein-synthesizing activity of cell-free systems

Changes in permeability of lysosomal and mitochondrial membranes were studied under different temperatures at which cryoextracts from organelles inhibit most distinctly the protein synthesizing activity of the cell-free system from the rat liver. It is found that mitochondria are more sensitive to the effect of low temperature effect than lysosomes. Overcooling of the mitochondria suspension to the temperature of the free water crystallization is shown to cause no release of the protein synthesis inhibitor. The inhibitor release from organelles occurs from the moment of crystallization and reaches its maximum at the eutectic temperatures of the freezing medium which is due to the injury effect of the complex of physicochemical factors on the membrane structures, occurring during the phase transition of the solvent.     

Study on the metastable zone width of ketoprofen

With increasing awareness for the need of pure enantiomer drugs, strong emphasis has been focused on the research of chiral drug separation. Compared with other separation methods, crystallization is a simple and economical method, and the metastable zone width (MSZW) is a very important factor for the entire crystallization process. In this paper, the effects of the metastable zones of (R,S)- and (S)-ketoprofen and a 0.94 mole fraction of (S)-ketoprofen in order to enhance the MSZW were studied. Four main factors were studied, namely, temperature, cooling rate, stirring rate, and volume ratio of mixed solvent (water/ethanol). Through the L9 fractional experiment design, it was observed that all samples' MSZWs would increase with an increase in cooling rate and decrease with an increase in the ethanol volume ratio and temperature. The ethanol ratio may have the strongest effect on the process and can greatly enhance the metastable zone, and the other three factors influence the MSZW only slightly. In conclusion, the these four factors for enhancing MSZW have been optimized: water-to-ethanol volume ratio, 1:0.6; temperature, 20 degrees C; stirring rate, 700 rpm; and cooling rate, 12.0 degrees C/h. All of these results will be helpful for the following chiral separation of ketoprofen by crystallization.     

The effect of temperature and solution pH on the nucleation of tetragonal lysozyme crystals.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.     

Control of nucleation in the crystallization of lysozyme.

This work investigates the influence of storage of lysozyme in solution on its crystallization. The crystallization of hen egg-white lysozyme exhibits a storage effect (aging) that depends on the length of time the lysozyme solution is stored, after dissolving from freeze-dried powder, before being brought to crystallization conditions. The number of crystals obtained increases, while their size decreases, as the solution ages. Observations suggest that this effect is due to the presence of fungi that multiply in the stored protein solution. This aging effect was used to control nucleation and determine the number and size of lysozyme crystals to be formed in a given sample.     

Application of Doehlert design to determine the combined effects of temperature, water activity and pH on conidial germination of Penicillium chrysogenum

AIMS: The influence of temperature, water activity and pH on the time necessary for germination of 90% of Penicillium chrysogenum conidia inoculated (T90) was determined. METHODS AND RESULTS: A new experimental device was developed for easy monitoring of the germination process. Experiments were carried out according to a Doehlert matrix at 11-31 degrees C, 0.86-0.98 water activity (a(w)) and pH 3.5-6.5. In these conditions, a second order polynomial relationship between T90 and the environmental factors was established for the different humectants used throughout this study (e.g. glycerol and sorbitol) with regression coefficients close to 0.97. CONCLUSIONS: For both humectants, the major effect of temperature and water activity on T90 was highlighted, whereas the effect of pH on T90 in these experimental conditions was not significant. The combined effect of temperature and water activity on T90 was also demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Both the experimental set-up and the Doehlert matrix were well suited to determine the influence of environmental factors on mould germination.     

Effect of tetracycline base crystallization on the conditions properties of the powders and drug forms obtained. The development of directed crystallization procedures for tetracycline base]

The study of the process of tetracycline base crystallization showed that with an increase in the rate of pH, temperature and mixed rotation changes, the specific surface of the crystalline precipitate increased with a simultaneous decrease in the bulk weight, looseness and volume density of the powder. The residual content of tetracycline in the mother solution decreased. The level of the effect of various parameters on the final results was different.     

Ice nucleation in nature: supercooling point (SCP) measurements and the role of heterogeneous nucleation

In biological systems, nucleation of ice from a supercooled aqueous solution is a stochastic process and always heterogeneous. The average time any solution may remain supercooled is determined only by the degree of supercooling and heterogeneous nucleation sites it encounters. Here we summarize the many and varied definitions of the so-called "supercooling point," also called the "temperature of crystallization" and the "nucleation temperature," and exhibit the natural, inherent width associated with this quantity. We describe a new method for accurate determination of the supercooling point, which takes into account the inherent statistical fluctuations of the value. We show further that many measurements on a single unchanging sample are required to make a statistically valid measure of the supercooling point. This raises an interesting difference in circumstances where such repeat measurements are inconvenient, or impossible, for example for live organism experiments. We also discuss the effect of solutes on this temperature of nucleation. Existing data appear to show that various solute species decrease the nucleation temperature somewhat more than the equivalent melting point depression. For non-ionic solutes the species appears not to be a significant factor whereas for ions the species does affect the level of decrease of the nucleation temperature.     

Interactions and phase behavior of a monoclonal antibody

Protein phase behavior is implicated in numerous aspects of downstream processing either by design, as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. An improved understanding of protein phase behavior is, therefore, important for developing rational design strategies for important process steps. This work explores the phase behavior of a monoclonal antibody (mAb), IDEC-152, which exhibits liquid-liquid separation, aggregation, gelation, and crystallization. A systematic study of numerous factors, including the effects of solution composition and pH, has been conducted to explore the phase behavior of this antibody. Phenomena observed include a significant dependence of the cloud point on the cation in sulfate salts and nonmonotonic trends in pH dependence. Additionally, conditions for crystallization of this mAb are reported for the first time. Protein-protein interactions, as determined from the osmotic second virial coefficient, are used to interpret the phase behavior.     

Evaluation of the stability of creatine in solution prepared from effervescent creatine formulations

The objectives of this study were to determine the cause of the crystallization in a large volume creatine supplement solution made from effervescent powders containing di-creatine citrate, and to characterize these crystals using thermal analyses and x-ray diffractometry. Creatine effervescent powders were dissolved in deionized water (pH 6.2) and stored both at room temperature (RT) (25°C) and refrigerated condition (4°C) over a period of 45 days. Creatine concentration was determined using high-performance liquid chromatography (HPLC). Intrinsic dissolution and saturated solubility of creatine, creatine monohydrate, and di-creatine citrate in water were determined and compared. Crystal growth was detected only in the refrigerated samples on the seventh day of storage. Differential Scanning Calorimetry (DSC) and x-ray diffraction (XRD) studies revealed that the crystals formed were of creatine monohydrate. Ninety percent creatine degradation was observed within 45 days for RT samples. However, at refrigerated condition this degradation was 80% within the same time period. The pH of the RT samples also increased from 3.6 to 4.5 during storage. No such increase was observed in the case of refrigerated samples. The intrinsic dissolution rate constants of the compounds decreased in the following order: dicreatine citrate>creatine>creatine monohydrate. In conclusion, di-creatine citrate used in effervescent formulation dissociates to creatine in aqueous solution and eventually crystallizes out as creatine monohydrate. Significant decrease in solubility and effect of pH contribute to this crystallization process.     

A new generation of sodium chloride porogen for tissue engineering

Porogen leaching is a widely used and simple technique for the creation of porous scaffolds in tissue engineering. Sodium chloride (NaCl) is the most commonly used porogen, but the current grinding and sieving methods generate salt particles with huge size variations and cannot generate porogens in the submicron size range. We have developed a facile method based on the principles of crystallization to precisely control salt crystal sizes down to a few microns within a narrow size distribution. The resulting NaCl crystal size could be controlled through the solution concentration, crystallization temperature, and crystallization time. A reduction in solution temperature, longer crystallization times, and an increase in salt concentration resulted in an increase in NaCl crystal sizes due to the lowered solubility of the salt solution. The nucleation and crystallization technique provides superior control over the resulting NaCl size distribution (13.78 ± 1.18 μm), whereas the traditional grinding and sieving methods produced NaCl porogens 13.89 ± 12.49 μm in size. The resulting NaCl porogens were used to fabricate scaffolds with increased interconnectivity, porous microchanneled scaffolds, and multiphasic vascular grafts. This new generation of salt porogen provides great freedom in designing versatile scaffolds for various tissue-engineering applications.     

Experimental determination of water equilibration rates in the hanging drop method of protein crystallization

The hanging drop method for protein crystallization consists of equilibrating a water droplet containing the protein and a precipitant against a reservoir solution at a higher precipitant concentration. The time for water equilibration--which affects the kinetics of crystallization--to reach 90% of completion is shown to vary between about 25 h and more than 25 days, depending on experimental conditions. Experiments were performed with three of the most widely used precipitants (ammonium sulfate, polyethylene glycol, 2-methyl-2,4-pentanediol), combining various parameters expected to affect the rate of water evaporation. The most dramatic effects were obtained when varying temperature and initial drop volume. A simple empirical equation for estimating the kinetics of water equilibration under given crystallization conditions is proposed.     

Kinetics of chiral resolution in stirred crystallization of D/L‐glutamic acid

Stirred crystallization of racemic (D/L)‐glutamic acid (Glu) in the presence of small amounts of L‐ or D‐lysine (Lys) was studied for the effect of transient chiral resolution by monitoring the time evolution of optical rotation and the concentration of the solution. The presence of a small amount of L‐ or D‐Lys retards the crystallization rate of the corresponding enantiomer of Glu in a chirally selective manner, giving rise to transient optical resolution of racemic Glu during crystallization. The optical rotation of the Glu solution was found to increase from zero to a value corresponding to an enantiomeric excess (ee) of 22–35% and subsequently decreases to zero over a period of many hours. During this process, the ee of the crystallized Gu is nearly 100% during the first 35 min and then it decreases slowly to zero. Our results indicate that the time at which the ee of the solution reaches its maximum and the maximum value of the ee show a nonlinear dependence on the initial mole fraction of the chiral impurity. The effect of the impurity is highly chirally selective, indicating “molecular recognition.” Chirality 11:343–348, 1999. © 1999 Wiley‐Liss, Inc.     

Freezing the effect of eutectic crystallization on biological membranes

Thylakoids from isolated spinach chloroplasts were frozen in the presence of various concentrations of inorganic and organic salts, amino acids and sugars and the kinetics of inactivation of cyclic photophosphorylation with phenazine methosulfate and of electron transport reactions were measured as a function of temperature.During freezing of membranes in the presence of neutral nontoxic compounds membrane damage did not occur until the eutectic temperature was reached. Then photophosphorylation became rapidly inactivated. With weakly membrane-toxic compounds there was a slow inactivation during freezing followed by rapid inactivation at the eutectic temperature. Freezing in the presence of strongly membrane-toxic compounds led to inactivation of photophosphorylation before the eutectic temperature was reached. The temperature at which eutectic crystallization occurred was dependent on the nature of the solutes present. The ratio between solute and membranes was also important: the lower the initial concentration of solutes added to membrane suspensions the lower the temperature at which eutectic solidification occurred. Some compounds such as mannitol crystallized gradually during the decrease in temperature; in this case inactivation of photophosphorylation took place parallel to the crystallization process.In contrast to photophosphorylation, electron transport reactions were not decreased during eutectic freezing in the presence of neutral membrane-protective compounds. Rather a stimulation of electron transport was observed. However, in the presence of inorganic salts or of sodium succinate, electron transport reactions were also inactivated in addition to photophosphorylation during eutectic solidification. This inactivation seems to be a salt effect and may not directly be related to the crystallization process. Various soluble enzymes and the Ca²⁺-dependent ATPase of thylakoids were not affected by eutectic crystallization.The results demonstrate that eutectic crystallization which may take place during freezing is a factor in membrane damage and has to be considered as a possible cause of membrane alterations in in vitro studies on freezing resistance.     

Influence of low-temperature nucleation on the crystallization process of poly(L-lactide)

The crystallization kinetics of poly(l-lactide), PLLA, is slow enough to allow a quasi-amorphous polymer to be obtained at low temperature simply by quenching from the melt. The PLLA crystallization process was followed by differential scanning calorimetry and optical microscopy after nucleation isothermal treatments at temperatures just below (53 degrees C) and just above (73 degrees C) the glass transition temperature. The crystallization exotherm shown in the heating thermograms shifts toward lower temperatures as the annealing time at 73 degrees C increases. The same effect is shown to a lesser extent when the sample nucleates at 53 degrees C, showing the ability to nucleate in the glassy state, already shown in other polymers. The shape of the DSC thermograms is modeled by using Avrami's theory and allows an estimation of the number of crystallization germs formed. The results of optical microscopy are converted to thermograms by evaluating the average gray level of the image recorded in transmission mode as a function of temperature and calculating its temperature derivative. The shape of such optical thermograms is quite similar to that of the DSC traces but shows some peculiarities after long nucleation treatments. Atomic force microscopy was used to analyze the crystal morphology and is an additional proof of the effect of nucleation in the glassy state. The crystalline morphology observed in samples crystallized after nucleation in the glassy state is qualitatively different from that of samples nucleated above the glass transition temperature, and the number of crystals seems to be much greater than what could be expected from the crystallization kinetics.     

Criteria for the crystallization of polysaccharides

The relationship between crystallization and the annealing process is well established in the synthetic polymer field. This relationship appears to have been somewhat neglected in studies on biopolymers. Results are presented to show the effect of both humidity and temperature in promoting structural changes in polysaccharide system. Three different polysaccharides have been used as examples of how crystallization may be speeded up by annealing at elevated temperatures.     

Effects of Addition of a Palmitic Sucrose Ester on Low-Trans-Fat Blends Crystallization in Bulk and in Oil-in-Water Emulsions

The effect of addition of a highly hydrophobic emulsifier, the palmitic sucrose ester P-170, on isothermal crystallization behavior of a high-melting fraction of milk fat (HMF) and its mixtures with 20% or 40% sunflower oil (SFO), both in bulk and in emulsion systems, were studied by nuclear magnetic resonance (NMR) and by time-resolved in situ small-angle synchrotron X-ray scattering (SAXS). NMR studies showed that the effect of P-170 on the overall isothermal crystallization kinetics in bulk phase could be either acceleration or delay. The effect was strongly dependent on supercooling. For HMF, P-170 retarded crystallization at temperatures above 29.0 ± 0.2 °C, while below that temperature, it accelerated the process. For the blends of HMF with 20% or 40% SFO, the temperature at which the behavior changed was 27.0 ± 0.2 or 26.0 ± 0.2 °C, respectively. In emulsion systems, however, the effect was always acceleration for all temperatures selected. With the aid of SAXS, it was possible to study early stage of crystallization during real time and therefore to improve our understanding of the mechanism of P-170 action. The different effects caused by P-170, as described by SFC curves, were strongly related to the effects of P-170 on fat polymorphism, specially to the value of the time interval of coexistence of the α and β′ forms. These events took place at the very beginning of crystallization and thus could not be described by the traditional X-ray techniques used in previous studies of similar systems.