A Direct Characterization Method of the Ice Morphology. Relationship Between Mean Crystals Size and Primary Drying Times of Freeze-Drying Processes

Abstract

In the freeze-drying process, the freezing step is one of the most important steps which determines the texture of the frozen material and, consequently, the final morphological characteristics of the freeze-dried material and its biological activity and its stability. As a matter of fact, the parameters of the freezing protocol have a direct effect on the pore size distribution and on the pore connectivity of the porous network of the freeze-dried matrix. Thus, the ice crystal morphology determines indirectly the mass and the heat transfer rates through the dry layer and, consequently, the freezing parameters have a strong influence on the total duration of the primary and secondary sublimation steps. The main objective of this study was to adapt and to develop a new optical direct microscopy method, based on the reflected flux differences, with episcopic axial lighting to characterize the structure of the different phases of a standard pharmaceutical matrix used for pharmaceutical proteins freeze-drying. First, the results obtained have been validated by another independent method, the scanning electron microscopy, carried out with freeze-dried samples. Finally, this technique has been principally used to investigate the effects of the freezing conditions on the ice crystal structure characterized by the distribution of the ice crystals mean sizes. Moreover, the influence of annealing treatment on ice crystal mean diameter and primary drying times has been also investigated.     

A Direct Characterization Method of the Ice Morphology. Relationship Between Mean Crystals Size and Primary Drying Times of Freeze-Drying Processes

In the freeze-drying process, the freezing step is one of the most important steps which determines the texture of the frozen material and, consequently, the final morphological characteristics of the freeze-dried material and its biological activity and its stability. As a matter of fact, the parameters of the freezing protocol have a direct effect on the pore size distribution and on the pore connectivity of the porous network of the freeze-dried matrix. Thus, the ice crystal morphology determines indirectly the mass and the heat transfer rates through the dry layer and, consequently, the freezing parameters have a strong influence on the total duration of the primary and secondary sublimation steps. The main objective of this study was to adapt and to develop a new optical direct microscopy method, based on the reflected flux differences, with episcopic axial lighting to characterize the structure of the different phases of a standard pharmaceutical matrix used for pharmaceutical proteins freeze-drying. First, the results obtained have been validated by another independent method, the scanning electron microscopy, carried out with freeze-dried samples. Finally, this technique has been principally used to investigate the effects of the freezing conditions on the ice crystal structure characterized by the distribution of the ice crystals mean sizes. Moreover, the influence of annealing treatment on ice crystal mean diameter and primary drying times has been also investigated.     

Ice crystal formation in the carbon nanotube suspension: A modelling approach

Freeze-dried porous solid foams were prepared from carbon nanotube (CNT) suspensions via either contact freezing with a heat exchanger or immersion freezing into a cryo-bath. Microstructures in the freeze-dried foam cast by ice crystals formed during the freezing step. It was found that domains of the carbon nanotubes in the freeze-dried solid foams were fairly well interconnected, and the microstructures were largely influenced by the freezing condition and freezing method. A mathematical model was proposed to simulate the observed thermal history during the freezing step; then theoretical predictions of the microstructural features were attempted. The simulated thermal history was in good agreement with experimental result. The resulting mean ice crystal sizes L* could be estimated from the calculated freezing front velocity (R) and the temperature gradient in the frozen zone (G). Interestingly, it was found that a correlation based on the power law (L*∝R^?0.2G^?0.2) was applicable to the present system for both the contact and immersion freezing methods. Though the fundamental ice crystallization phenomena were essentially the same for both freezing methods, actual temperature distribution and movement of the freezing front through the whole freezing bulk could control the morphology of ice crystal in the frozen matrix. In other words, good control of thermal flow in the freezing system would greatly contribute to rational design of microstructures of the CNT foam.     

Freezing step controls the mannitol phase composition heterogeneity

Freeze-dried samples were prepared from D-mannitol solution by selected freezing conditions. Crystalline structures of prepared samples were determined by XRD analysis, and distributions of the various crystal structures of mannitol were obtained for each sample. It was found that the amount of each polymorph was quite well correlated to the freezing conditions, namely the ice crystal nucleation temperature and the cooling rate. In case of samples prepared at fast cooling rates, the samples where the ice crystals nucleated at higher temperature contained much more stable form than the samples nucleated at lower temperature. Besides, the samples prepared at slow cooling rates predominantly contained stable crystalline forms despite of the variation of nucleation temperatures. Moreover, the experimental results also indicated that the various polymorphs were also distributed vertically through the sample along the direction of the heat flux during freezing. The tendency of the polymorph distribution through the freeze-dried cake was also elucidated by using the simulated temperature profiles during freezing. Thus, the profiles of mannitol polymorphs after the freezing derived from the temperature distributions could predict the global tendency of polymorphism behaviour, and, consequently, would be useful to achieve quality control of freeze-dried formulations.     

Effect of Ultrasonically Induced Nucleation on the Drying Kinetics and Physical Properties of Freeze-Dried Strawberry

Power ultrasound has proved to be very useful in controlling crystallization processes since sonication can enhance both the nucleation rate and crystal growth rate by producing fresh and/or more nucleation sites. Therefore, in this study, power ultrasound was applied to control the freezing step of freeze-dried strawberry. The results showed that when power ultrasound was applied at different temperatures, it increased the nucleation temperature and shortened the characteristic freezing time. The application of power ultrasound in the freezing step increased the drying time in subsequently freeze-dried strawberry samples. This longer drying time was found to be due to increased resistance to moisture diffusion due to the formation of a network of small pores caused by sublimation of small ice crystals induced by the power ultrasound. Scanning electron microscopic images revealed that the freeze-dried sample frozen by ultrasound-assisted freezing (UAFD) had finer cellular structure compared to those frozen in other freezing conditions. UAFD samples had better textural hardness, while the rehydration capacity was lower compared to those of NRFD and NIFD samples.     

A Review on Freeze Drying of Drugs with tert-Butanol (TBA) + Water Systems: Characteristics,Advantages, Drawbacks

Organic cosolvent systems have been evaluated for their potential and increasing use for freeze drying of solutions to produce stabilized powders of marketed pharmaceutical products. The formulations most often investigated include tert-butanol (TBA) + water mixtures. These organic cosolvent systems present many interesting advantages: high freezing temperatures, very short sublimation time, low sublimation enthalpies, high equilibrium vapor/solid pressure values, and low toxicity.

Thus, some main characteristics of water + TBA systems have been reviewed, especially regarding its interesting thermodynamic properties (sublimation enthalpy, equilibrium vapor pressure), impact of freezing conditions on morphological properties of frozen formulations (nucleation, crystal size and shape), influence of operating parameters (total pressure, temperature) on sublimation times, and organic cosolvent and water residual contents.

The crystal morphology of frozen formulations prepared with TBA revealed unexpected results compared with the results reported in the literature for water-based formulations in terms of the complex relationships between freezing rates, supercooling, nucleation temperatures, and solvent crystal morphology (size and shape).

It has been proved that optimum freeze-drying cycles are established by simultaneously taking into account the impact of formulation variables, especially the tert-butanol content, and classical freeze-drying variables during the freezing step (nucleation temperatures, freezing rates) and the sublimation step (shelf temperature, total pressure) to maximize the drying rates and to minimize the residual solvent levels while preserving the main quality attributes of the freeze-dried powder.     

Simulation of freezing step in vial lyophilization using finite element method

Determination of heat transfer and temperature profile during freezing step is fundamental to predict the final structure of a lyophilized product. The aim of this study was to develop and optimize a dynamic model based on finite element method for simulation of freezing step in order to study final product morphology in both vertical and radial directions. Different factors have been taken into account: chamber pressure, shelf temperature, vial shape, initial solution temperature, nucleation temperature and phase changes. The dynamic axisymmetric model proposed could simulate temperature of each point in the vial and position of liquid-solid interface, without necessity of fitting parameters or questionable assumptions. In addition, the model was extended to predict the average crystal size in each element and the influence of different factors was examined.     

Cell-culture compatible silk fibroin scaffolds concomitantly patterned by freezing conditions and salt concentration

The morphology of freeze-dried silk fibroin 3D-scaffolds was modified by varying both the NaCl concentration and the freezing temperature of the silk fibroin solution prior to lyophilization. Scanning electron micrographs showed that slow freezing at −22 °C generated sponge-like interconnected porous networks, whereas fast freezing at −73 °C formed stacked leaflet structures. The presence of millimolar NaCl (50–250 mM) increased the porosity of the scaffolds and generated small outgrowths at their surface, depending on the freezing regime. Our results suggest that the morphological differences seen between the materials likely depend on ice and NaCl hydrate crystal nucleation and growth mechanisms. Infrared spectroscopy and X-ray diffraction analyses revealed that the salt concentration and freezing conditions induced no structural changes in fibroin. The seeding of P19 embryonic carcinoma cells showed that the presence of salt and freezing conditions influenced the cell distribution into the scaffolds, with salt addition increasing the access of cells to deeper regions.     

单向冷冻过程溶液中冰晶界面的竞争现象

通过对溶液冷冻过程冰晶界面发展变化的实验观察,定义了界面发展过程的3个阶段,分析各个阶段的现象特征,并从界面区浓度分布探析界面规则形态的物理本质。界面的发展过程中,会因为来自内部或者外界的扰动,使界面局部区域产生不均匀性,导致晶枝间的竞争现象,而决定这些竞争现象及冰晶生长的驱动因素是界面附近的过冷度分布。竞争现象机理的分析,有助于更清晰地了解生物材料在冷冻过程中的结冰特性。     

药品冷冻干燥过程的退火机理分析

运用热力学和动力学理论分析了药品冷冻干燥过程中的退火操作改变冻结药品结构的机理。通过对化学势随晶粒曲率半径变化、重结晶过程中冰晶半径随时间变化、非晶态基质的粘度随温度变化几个关系式的分析,得出退火温度必须要高于冻结药品的玻璃化转变温度;最佳退火持续时间与重结晶的速率紧密相关,且是退火温度的函数;退火后再冷却过程的冷却速率不影响升华干燥速率等结论。     

The effect of variation in pore structure on the frost resistance of porous materials

Processes of freezing of water and melting of ice were studied using samples of hardened suspensions of plaster-of-paris with different mean pore sizes and constant porosities. Changes in the structure of the material were analyzed by measurement of the thermal and volume changes during cooling and heating cycles, and by the measurement of pore-size distribution and the changes of strength after the freezing cycles. It was shown that changes of volume within a temperature limit T=±20°C depend on the mean pore size and on the degree of water saturation. The disruption of the hardened plaster-of-paris structure was evaluated by determining the decrease in strength, if the nucleation of the ice crystals occurs during supercooling and the rate of their growth is greater than the rate of the amount of water forced out of the pores. The experimental observations support Power's theory concerning the frost resistance of porous material.     

Freeze Decontamination Process: Modeling in a Simplified Case of Completely Mixed Aqueous Phase and Observations with Ultrasonic Agitation in the Liquid during Freezing

Abstract

Correlations among variables of the freeze decontamination process have not been developed to precisely predict experimental observations of purified solid ice out of a given aqueous solution on a macroscale. A lack of such information has been one of the main reasons for the very small practical application of the process so far. A simplified model has been presented in an effort to take into account the inhomogeneity of the solid phase. Pure ice spikes scattered on a cold surface have been supposed to grow opposite to the direction of heat dissipation, leaving unfrozen liquid channels among them along their length. The ice phase has thus been supposed to consist of pure ice spikes and a quantity of liquid entrapped among them. The liquid at the freezing front and in bulk is supposed to be completely mixed. Volume changes on freezing have also been taken into account. The model has been compared with experimental observations on a specially fabricated apparatus. Temperatures of a cold freezing surface have been continuously lowered while following a ?0.0017 K/s gradient during experiments. An ultrasonic mixing technique has been applied to the liquid during freezing to eliminate impurity (cesium nitrate) concentration gradients in the liquid. A comparison of the model with experimental observations has been made with the help of a decontamination factor (DF) [concentration of impurity in the initial liquid/ concentration of impurity in the frozen mass (after it has been melted)] versus V/V ₀ (volume of unfrozen liquid left at any point during freezing/volume of initial liquid taken) plots. Although a scatter in the experimental results has been observed, there was broad agreement with the model trend. The average value of DF has been as high as 46. The investigation has highlighted the importance of the factors that determine the fraction of pure ice spikes in the growing solid at any point during freezing.     

Three-Dimensional Measurement of Ice Crystals in Frozen Materials by Near-Infrared Imaging Spectroscopy

A novel technique was developed to recognize ice crystals in biological materials and to analyze their three-dimensional morphology using a Cryogenic Micro-Slicer Spectral Imaging System with a micro-slicer unit and a near-infrared spectral imaging unit. Consecutive cross-sections of a frozen sample were exposed by the multi-slicing operations with a minimum thickness of 1 µm, and their images were taken by the imaging unit. Spectroscopic analysis using a near-infrared spectrum meter showed an absorption peak at 1460 nm for pure water. Based on the observations of the absorption band of ice crystals in the wavelength range of 1450–1570 nm and its peak at 1495 nm, a commodity-type bandpass filter with a central wavelength of 1500 nm was adopted to identify ice crystals in near-infrared images. The absorption peak of water exhibited a tendency to move toward longer wavelengths with decreasing sample temperature from 25 °C to ?15 °C. The filtered images of ice crystals in frozen samples were darker than the other components at the peak wavelength of ice crystals. The three-dimensional reconstructed morphology of ice crystals revealed that they were formed along the direction of heat transfer while freezing. The proposed method provides a novel tool to investigate the effects of freezing conditions on the size, morphology and distribution of ice crystals.     

冷冻保存中影响胞内冰生长的因素

细胞胞内冰的形成会导致严重的细胞损伤从而导致低温贮存中的诸多问题。以蚕豆为研究对象,用细胞松弛素B溶解细胞骨架,使用低温显微系统在不同的冷却速率下进行冷冻实验。实验结果表明,使用细胞松弛素B处理过的细胞在冷冻过程中结晶温度更高,结晶时间更短,但细胞骨架对胞内冰的生长过程影响较小。外界条件起着关键作用,接种冰晶影响细胞内冰晶的形成温度及冰晶的生长速率。最后,通过光强度图对细胞的损伤程度进行了分析。     

The effect of shear on the crystallization of cocoa butter

This paper examines the effect of shear on the crystallization of cocoa butter using a combination of three different experimental techniques and a single crystallization temperature of 20°C. Rheological measurements were carried out to study the effect of a shear step on the crystallization kinetics of the fat. Without a shear step, little rheological change was observed at 20°C; however, with the application of a shear step the onset of significant rheological change occurred and was strongly influenced by the magnitude of the shear step. Detailed crystallographic measurements could be made with in situ X-ray experiments during flow-induced crystallization. The imposition of continuous shear changed both crystal polymorphic structure and crystallization kinetics in a systematic way. Finally, optical measurements were used to follow changes in crystal morphology as a consequence of continuous shear. These results revealed the form and kinetics of crystal growth. In general the results complemented each other, and an overall picture of the way shear influenced cocoa butter growth could be formed. The observations could be the basis for a future mathematical model of growth kinetics and provide insight into the way shear influences crystallization kinetics, morphology, and polymorphic structure.     

Influence of Freezing Temperature on Product Parameters of Solid Dosage Forms Prepared via the Freeze‐Casting Technique

The freeze‐casting technique was used to formulate porous solid bodies containing theophylline as an active pharmaceutical ingredient, potato starch as a filler and citric acid or saccharose as binding agents. Aqueous suspensions of the ingredients were frozen at three different temperatures. The aim of this work was to investigate the influence of the freezing temperature on the ice crystal growth rate of the suspensions and the resulting porosity of the freeze‐casted samples. The impact of the freezing temperature on the solidification of the suspensions was analyzed via contact angle measurements. The rate of ice crystal growth was expressed as an overall linear growth rate. The porosity of the freeze‐casted bodies was determined by high‐pressure mercury porosimetry. A close correlation was found between the freezing temperature and the investigated product parameters. Lower cooling temperatures resulted in higher final porosities. The lowest temperature, at which final product properties could be regulated by varying the cooling regimes, was –30 °C. The influence of the freezing temperature and the impact of the additives were not remarkable at a freezing temperature lower than –30 °C. Therefore, it can be concluded that the optimum freezing temperature of the suspensions investigated in this study is in the temperature range between –20 °C and –30 °C.     

Desalination by natural freezing

This paper presents a technique for desalination by natural freezing that can be used in relatively warm climates with ambient temperatures above freezing. The reasons for the freezing of water are discussed and theoretical calculations are given to predict the time required to reach the freezing temperature. Experimental results are given which show that the technique works. The melted ice water from a single freezing without a wash step has three to six times less salt than the feed. Possible suitable locations, a preliminary economic analysis, and methods of increasing the product purity by washing the ice are discussed.     

真空探针冷冻和复温性能实验测试及数值模拟

针对现有的冷热复合治疗探针中存在的问题,设计了直径3 mm的真空探针,用电阻加热和真空层相结合的方法解决探针非工作段的低温问题,以液氮冷冻和电阻加热的方式实现探针的冷热交替过程。分别在空气、蒸馏水和离体猪肝中进行探针的性能测试实验。发现电加热与真空层结合可以提高探针非工作段的温度;在蒸馏水中探针形成轴向长度为3.6 cm,径向长度为1.8 cm的冰球;在离体猪肝中形成轴向冻结直径约为3.6 cm,化冻直径为1.2 cm的区域。使用数值计算的方法计算探针的有效治疗范围,形象直观地展示组织在冷冻和复温过程中的温度场分布,为临床手术提供数据支持。从整体效果看,探针有良好的冷冻和复温性能,促进冷热复合治疗的进一步发展。     

Freeze-drying of ovalbumin-loaded carboxymethyl chitosan nanocapsules: Impact of freezing and annealing procedures on physicochemical properties of the formulation during dried storage

Nanocapsules were prepared from 6-O-carboxymethyl chitosan, ovalbumin, and calcium chloride as a cross-linker agent. Cryoprotectants were added to the formulation to protect these carriers during freezing and desiccation steps. The nanocapsules showed acceptable stability with regard to the residual humidity and cake appearance. Regarding the stability, particle diameter, and polydispersity index increased over the storage time, especially for formulations stored at room temperature. For all studied lyophilization protocols exclusively δ-mannitol was formed, and the addition of an annealing step maximized mannitol crystallization. The decrease of the pore surface area for annealed formulations resulted of structural changes other than Ostwald ripening.