Physico-chemical Processes that Occur During Freezing

Physico-chemical Processes that Occur During Freezing 1. Introduction Lyophilization respectively freeze-drying is an important and well-established process to improve the long-term stability of labile drugs, especially therapeutic proteins.[1] About 50% of the currently marketed biopharmaceuticals are lyophilized, representing the most common formulation strategy.[2] In the freeze-dried solid state chemical or physical degradation reactions are inhibited or sufficiently decelerated, resulting in an improved long-term stability.[3] Besides the advantage of better stability, lyophilized formulations also provide easy handling during shipping and storage. [1] A traditional lyophilization cycle consists of three steps; freezing, primary drying and secondary drying.[1] During the freezing step, the liquid formulation is cooled until ice starts to nucleate, which is followed by ice growth, resulting in a separation of most of the water into ice crystals from a matrix of glassy and/or crystalline solutes.[4-5] During primary drying, the crystalline ice formed during freezing is removed by sublimation. Therefore, the chamber pressure is reduced well below the vapor pressure of ice and the shelf temperature is raised to supply the heat removed by ice sublimation.[6] At the completion of primary drying, the product can still contain approximately 15% to 20% of unfrozen water, which is desorbed during the secondary drying stage, usually at elevated temperature and low pressure, to finally achieve the desired low moisture content.[7] In general, lyophilization is a very time- and energy-intensive drying process.[8]   Typically, freezing is over within a few hours while drying often requires days. Within the drying phase, secondary drying is short (~hours) compared to primary drying (~days).[1, 4] Therefore, lyophilization cycle development has typically focused on optimizing the primary drying step, i.e., shortening the primary drying time by adjusting the shelf temperature and/or chamber pressure without influencing product quality.[5, 9] Although, freezing is one of the most critical stages during lyophilization, the importance of the freezing process has rather been neglected in the past.[10]   The freezing step is of paramount importance. At first, freezing itself is the major desiccation step in lyophilization [6] as solvent water is removed from the liquid formulation in the form of a pure solid ice phase, leading to a dramatic concentration of the solutes.[11-12] Moreover, the kinetics of ice nucleation and crystal growth determine the physical state and morphology of the frozen cake and consequently the final properties of the freeze-dried product.[11-13] Ice morphology is directly correlated with the rate of sublimation in primary and secondary drying.[14] In addition, freezing is a critical step with regard to the biological activity and stability of the active pharmaceutical ingredients (API), especially pharmaceutical proteins.[1] While simple in concept, the freezing process is presumably the most complex but also the most important step in the lyophilization process.[10] To meet this challenge, a thorough understanding of the physico-chemical processes, which occur during freezing, is required. Moreover, in order to optimize the freeze drying process and product quality, it is vital to control the freezing step, which is challenging because of the random nature of ice nucleation. However, several approaches have been developed to trigger ice nucleation during freezing. The purpose of this review is to provide the reader with an awareness of the importance but also complexity of the physico-chemical processes that occur during freezing. In addition, currently available freezing techniques are summarized and an attempt is made to address the consequences of the freezing procedure on process performance and product quality. A special focus is set on the critical factors that influence protein stability. Understanding and controlling the freezing step in lyophilization will lead to optimized, more efficient lyophilization cycles and products with an improved stability. 2. Physico-chemical fundamentals of freezing The freezing process first involves the cooling of the solution until ice nucleation occurs. Then ice crystals begin to grow at a certain rate, resulting in freeze concentration of the solution, a process that can result in both crystalline and amorphous solids, or in mixtures.[11] In general, freezing is defined as the process of ice crystallization from supercooled water.[15] The following section summarizes the physico-chemical fundamentals of freezing. At first, the distinction between cooling rate and freezing rate should be emphasized. The cooling rate is defined as the rate at which a solution is cooled, whereas the freezing rate is referred to as the rate of postnucleation ice crystal growth, which is largely determined by the amount of supercooling prior to nucleation.[16-17] Thus, the freezing rate of a formulation is not necessarily related to its cooling rate.[18] 2.1 Freezing phenomena: supercooling, ice nucleation and ice crystal formation In order to review the physico-chemical processes that occur during freezing of pure water, the relationship between time and temperature during freezing is displayed in figure 1. When pure water is cooled at atmospheric pressure, it does not freeze spontaneously at its equilibrium freezing point (0 °C).[19] This retention of the liquid state below the equilibrium freezing point of the solution is termed as â€Å"supercooling†.[19] Supercooling (represented by line A) always occurs during freezing and is often in the range of 10 to 15 °C or more.[12, 18] The degree of supercooling is defined as the difference between the equilibrium ice formation temperature and the actual temperature at which ice crystals first form and depends on the solution properties and process conditions.[1, 6, 11, 20] As discussed later, it is necessary to distinguish between â€Å"global supercooling†, in which the entire liquid volume exhibits a similar level of supercooling, and â€Å"lo cal supercooling†, in which only a small volume of the liquid is supercooled.[14] Supercooling is a non-equilibrium, meta-stable state, which is similar to an activation energy necessary for the nucleation process.[21] Due to density fluctuations from Brownian motion in the supercooled liquid water, water molecules form clusters with relatively long-living hydrogen bonds [22] almost with the same molecular arrangement as in ice crystals.[11, 15] As this process is energetically unfavorable, these clusters break up rapidly.[15] The probability for these nuclei to grow in both number and size is more pronounced at lowered temperature.[15] Once the critical mass of nuclei is reached, ice crystallization occurs rapidly in the entire system (point B).[15, 21-22]   The limiting nucleation temperature of water appears to be at about -40 °C, referred to as the â€Å"homogeneous nucleation temperature†, at which the pure water sample will contain at least one spontaneously f ormed active water nucleus, capable of initiating ice crystal growth.[11] However, in all pharmaceutical solutions and even in sterile-filtered water for injection, the nucleation observed is â€Å"heterogeneous nucleation†, meaning that ice-like clusters are formed via adsorption of layers of water on â€Å"foreign impurities†.[6, 11] Such â€Å"foreign impurities† may be the surface of the container, particulate contaminants present in the water, or even sites on large molecules such as proteins.[23-24] Primary nucleation is defined as the initial, heterogeneous ice nucleation event and it is rapidly followed by secondary nucleation, which moves with a front velocity on the order of mm/s through the solution. [14, 25] Often secondary nucleation is simply referred to as ice crystallization, and the front velocity is sometime referred to as the crystallization linear velocity.[14] Once stable ice crystals are formed, ice crystal growth proceeds by the addition of molecules to the interface.[22] However, only a fraction of the freezable water freezes immediately, as the supercooled water can absorb only 15cal/g of the 79cal/g of heat given off by the exothermic ice formation.[12, 22] Therefore, once crystallization begins, the product temperature rises rapidly to near the equilibrium freezing point.[12, 26] After the initial ice network has formed (point C), additional heat is removed from the solution by further cooling and the remaining water freezes when the previously formed ice crystals grow.[12] The ice crystal growth is controlled by the latent heat release and the cooling rate, to which the sample is exposed to.[22] The freezing time is defined as the time from the completed ice nucleation to the removal of latent heat (from point C to point D). The temperature drops when the freezing of the sample is completed (point E).[21] The number of ice nuclei formed, the rate of ice growth and thus the ice crystals` size depend on the degree of supercooling.[14, 20] The higher the degree of supercooling, the higher is the nucleation rate and the faster is the effective rate of freezing, resulting in a high number of small ice crystals. In contrast, at a low degree of supercooling, one observes a low number of large ice crystals.[14, 19] The rate of ice crystal growth can be expressed as a function of the degree of supercooling.[23]   For example for water for injection, showing a degree of supercooling of 10 °C +/- 3 °C, an ice crystal growth rate of about   5.2cm/s results.[23] In general, a slower cooling rate leads to a faster freezing rate and vice versa. Thus, in case of cooling rate versus freezing rate it has to be kept in mind â€Å"slow is fast and fast is slow†. Nevertheless, one has to distinguish between the two basic freezing mechanisms. When global supercooling occurs, which is typically the case for shelf-ramped freezing, the entire liquid volume achieves a similar level of supercooling and solidification progresses through the already nucleated volume.[12, 14] In contrast, directional solidification occurs when a small volume is supercooled, which is the case for high cooling rates, e.g. with nitrogen immersion. Here, the nucleation and solidification front are in close proximity in space and time and move further into non-nucleated solution. In this case, a faster cooling rate will lead to a faster freezing rate.[12, 14] Moreover, as ice nucleation is a stochastically event [6, 18], ice nucleation and in consequence ice crystal size distribution will differ from vial to vial resulting in a huge sample heterogeneity within one batch.[6, 14, 27] In addition, during freezing the growth of ice crystals within one vial can also be heterogeneous, influencing intra-vial uniformity.[5] Up to now, 10 polymorphic forms of ice are described. However, at temperatures and pressures typical for lyophilization, the stable crystal structure of ice is limited to the hexagonal type, in which each oxygen atom is tetrahedrally surrounded by four other oxygen atoms.[23] The fact that the ice crystal morphology is a unique function of the nucleation temperature was first reported by Tammann in 1925.[28] He found that frozen samples appeared dendritic at low supercoolings and like â€Å"crystal filaments† at high supercooling. In general, three different types of growth of ice crystals around nuclei can be observed in solution[15]: i) if the water molecules are given sufficient time, they arrange themselves regularly into hexagonal crystals, called dendrites; ii) if the water molecules are incorporated randomly into the crystal at a fast rate, â€Å"irregular dendrites† or axial columns that originate from the center of crystallization are formed; iii) at higher coo ling rates, many ice spears originate from the center of crystallization without side branches, referred to as spherulites. However, the ice morphology depends not only on the degree of supercooling but also on the freezing mechanism. It is reported that â€Å"global solidification† creates spherulitic ice crystals, whereas â€Å"directional solidification† results in directional lamellar morphologies with connected pores.[12, 14] While some solutes will have almost no effect on ice structure, other solutes can affect not only the ice structure but also its physical properties.[19] Especially at high concentrations, the presence of solutes will result in a depression of the freezing point of the solution based on Raoults`s Law and in a faster ice nucleation because of the promotion of heterogeneous nucleation, leading to a enormously lowered degree of supercooling.[21] 2.2 Crystallization and vitrification of solutes The hexagonal structure of ice is of paramount importance in lyophilization of pharmaceutical formulations, because most solutes cannot fit in the dense structure of the hexagonal ice, when ice forms.[23] Consequently, the concentration of the solute constituents of the formulation is increased in the interstitial region between the growing ice crystals, which is referred to as â€Å"cryoconcentration†.[11-12] If this separation would not take place, a solid solution would be formed, with a greatly reduced vapor pressure and the formulation cannot be lyophilized.[23] The total solute concentration increases rapidly and is only a function of the temperature and independent of the initial concentration.[4] For example, for an isotonic saline solution a 20-fold concentration increase is reported when cooled to -10 °C and all other components in a mixture will show similar concentration increases.[4] Upon further cooling the solution will increase to a critical concentration, ab ove which the concentrated solution will either undergo eutectic freezing or vitrification.[7] A simple behavior is crystallization of solutes from cryoconcentrated solution to form an eutectic mixture.[19] For example, mannitol, glycine, sodium chloride and phosphate buffers are known to crystallize upon freezing, if present as the major component.[12] When such a solution is cooled, pure ice crystals will form first. Two phases are present, ice and freeze-concentrated solution. The composition is determined via the equilibrium freezing curve of water in the presence of the solute (figure 2). The system will then follow the specific equilibrium freezing curve, as the solute content increases because more pure water is removed via ice formation. At a certain temperature, the eutectic melting temperature (Teu), and at a certain solute concentration (Ceu), the freezing curve will meet the solubility curve. Here, the freeze concentrate is saturated and eutectic freezing, which means solute crystallization, will occur.[7, 19] Only below Teu, which is defined as the lowest temperat ure at which the solute remains a liquid the system is completely solidified.[19] The Teu and Ceu are independent of the initial concentration of the solution.[7] In general, the lower the solubility of a given solute in water, the higher is the Teu.[19] For multicomponent systems, a general rule is that the crystallization of any component is influenced, i.e. retarded, by other components.[11] In practice, analogous to the supercooling of water, only a few solutes will spontaneously crystallize at Teu.[11] Such delayed crystallization of solutes from a freezing solution is termed supersaturation and can lead to an even more extreme freeze concentration.[11] Moreover, supersaturation can inhibit complete crystallization leading to a meta-stable glass formation, e.g. of mannitol.[12, 23] In addition, it is also possible that crystalline states exist in a mixture of different polymorphs or as hydrates.[11] For example, mannitol can exist in the form of several polymorphs (a, b and d) und under certain processing conditions, it can crystallize as a monohydrate.[11] The phase behavior is totally different for polyhydroxy compounds like sucrose, which do not crystallize at all from a freezing solution in real time.[11] The fact that sucrose does not crystallize during freeze-concentration is an indication of its extremely complex crystal structure.[11] The interactions between sugar -OH groups and those between sugar -OH groups and water molecules are closely similar in energy and configuration, resulting in very low nucleation probabilities.[11] In this case, water continues to freeze beyond the eutectic melting temperature and the solution becomes increasingly supersaturated and viscous.[11] The increasing viscosity slows down ice crystallization, until at some characteristic temperature no further freezing occurs.[11] This is called glassification or vitrification.[18]   The temperature at which the maximal freeze-concentration (Cg`) occurs is referred to as the glass transition temperature Tg`.[11, 29] This point is at the intersection of t he freezing point depression curve and the glass transition or isoviscosity curve, described in the â€Å"supplemented phase diagram† [30] or â€Å"state diagram† (figure 2).[11] Tg ´ is the point on the glass transition curve, representing a reversible change between viscous, rubber-like liquid and rigid, glass system.[19] In the region of the glass transition, the viscosity of the freeze concentrate changes about four orders of magnitude over a temperature range of a few degrees.[19] Tg` depends on the composition of the solution, but is independent of the initial concentration.[4, 11, 27]   For example, for the maximally freeze concentration of sucrose a concentration of 72-73% is reported.[31] In addition to Tg` the collapse temperature (Tc) of a product is used to define more precisely the temperature at which a structural loss of the product will occur. In general Tc is several degrees higher than Tg`, as the high viscosity of the sample close to Tg` will pre vent .[10] The glassy state is a solid solution of concentrated solutes and unfrozen, amorphous water. It is thermodynamically unstable with respect to the crystal form, but the viscosity is high enough, in the order of 1014 Pa*s, that any motion is in the order of mm/year.[4, 11, 29] The important difference between eutectic crystallization and vitrification is that for crystalline material, the interstitial between the ice crystal matrix consists of an intimate mixture of small crystals of ice and solute, whereas for amorphous solutes, the interstitial region consists of solid solution and unfrozen, amorphous water.[19, 23] Thus, for crystalline material nearly all water is frozen and can easily be removed during primary drying without requiring secondary drying.[19] However, for amorphous solutes, about 20% of unfrozen water is associated in the solid solution, which must be removed by a diffusion process during secondary drying.[19] Moreover, the Teu for crystalline material or the Tg` respectively Tc for amorphous material define the maximal allowable product temperature during primary drying.[19] Eutectic melting temperatures are relatively high compared to glass transition temperatures, allowing a higher product temperature during primary drying, which resu lts in more efficient drying processes.[19] If the product temperature exceeds this critical temperature crystalline melting or amorphous collapse will occur, resulting in a loss of structure in the freeze-dried product, which is termed â€Å"cake collapse†.[11, 19] 2.3 Phase separation and other types of freezing behavior A characteristic property of multicomponent aqueous solutions, especially when at least one component is a polymer, is the occurrence of a liquid-liquid phase separation during freezing into two liquid equilibrium phases, which are enriched in one component.[11, 19] This phase separation behavior has been reported for aqueous solutions of polymers such as PEG/dextran or PVP/dextran but is also reported for proteins and excipients.[32-33] When a critical concentration of the solutes is reached, the enthalpically unfavorable interactions between the solutes exceed the favorable entropy of a solution with complete miscibility.[34] Another proposed explanation is that solutes have different effects on the structure of water, leading to phase separation.[35] Besides the separation into two amorphous phases, two other types of phase separation are stated in literature; crystallization of amorphous solids and amorphization from crystalline solids.[18] Crystallization of amorphous solids often occurs when metastable glasses are formed during freezing. In this case, e.g. upon extremely fast cooling, a compound that normally would crystallize during slower freezing is entrapped as an amorphous, metastable glass in the freeze-concentrate.[12, 23] However, with subsequent heating above Tg`, it will undergo crystallization, which is the basis for annealing during freeze-drying (see 3.3).[19] Without annealing, the metastable glass can crystallize spontaneously out of the amorphous phase during drying or storage.[18] Amorphization from crystalline solids, that can be buffer components or stabilizers, predominantly occurs during the drying step and not during the freezing step.[18, 36]   Additionally, lyotropic liquid crystals, which have the degree of order between amorphous and crystalline, are reported to form as a result of freeze-concentration. However, their influence on critical quality attributes of the lyophilized product are not clarified.[19] Moreover, clathrates, also termed gas hydrates, are known to form, especially in the presence of non-aqueous co-solvents, when the solute alters the structure of the water.[23] 3. Modifications of the freezing step As aforementioned, the ice nucleation temperature defines the size, number and morphology of the ice crystals formed during freezing. Therefore, the statistical nature of ice nucleation poses a major challenge for process control during lyophilization. This highlights the importance of a controlled, reproducible and homogeneous freezing process. Several methods have been developed in order to control and optimize the freezing step. Some of them only intend to influence ice nucleation by modifying the cooling rate. Others just statistically increase the mean nucleation temperature, while a few allow a true control of the nucleation at the desired nucleation temperature. 3.1 Shelf-ramped freezing Shelf-ramped freezing is the most often employed, conventional freezing condition in lyophilization.[37] Here, at first, the filled vials are placed on the shelves of the lyophilizer and the shelf temperature is then decreased linearly (0.1 °C/min up to 5 °C/min, depending on the capacity of the lyophilizer) with time.[37-38] As both water and ice have low thermal conductivities and large heat capacities and as the thermal conductivity between vials and shelf is limited, the shelf-ramped cooling rate is by nature slow.[11] In order to ensure the complete solidification of the samples, the samples must be cooled below Tg` for amorphous material respectively below Teu for crystalline material. Traditionally, many lyophilization cycles use a final shelf temperature of -50 °C or lower, as this was the maximal cooling temperature of the freeze-drier.[7] Nowadays, it is suggested to use a final shelf temperature of -40 °C if the Tg` or Teu is higher than -38 °C or to use a temper ature of 2 °C less than Tg` and Teu.[1] Moreover, complete solidification requires significant time.[11] In general, the time for complete solidification depends on the fill volume; the larger the fill volume the more time is required for complete solidification.[11] Tang et al.[1]   suggest that the final shelf temperature should be held for 1 h for samples with a fill depth of less than or equal to 1 cm or 2 h for samples with a fill depth of greater than 1 cm. Moreover, fill depth of greater than 2 cm should be avoided, but if required, the holding time should be increased proportionately. In order to obtain a more homogeneous freezing, often the vials are equilibrated for about 15 to 30 min at a lowered shelf temperature (5 °C 10 °C) before the shelf temperature is linearly decreased.[1] Here, either the vials are directly loaded on the cooled shelves or the vials are loaded at ambient temperature and the shelf temperature is decreased to the hold temperature. [1, 5, 9] Another modification of the shelf-ramped freezing is the two-step freezing, where a â€Å"supercooling holding† is applied.(7) Here, the shelf temperature is decreased from room temperature or from a preset lowered shelf temperature to about -5 to -10 °C for 30 to 60min hold. This leads to a more homogenous supercooling state across the total fill volume.[1, 5] When the shelf temperature is then further decreased, relatively homogeneous ice formation is observed.[5] In general, shelf-ramped frozen samples show a high degree of supercooling but when the nucleation temperature is reached, ice crystal growth proceeds extremely fast, resulting in many small ice crystals.[9, 39] However, the ice nucleation cannot be directly controlled when shelf-ramped freezing is applied and is therefore quite random.[4] Thus, one drawback of shelf-ramped freezing is that different vials may become subject to different degrees of supercooling, typically about +/- 3 °C about the mean.[4] This results in a great variability in product quality and process performance.[4] Moreover, with the shelf-ramped freezing method it is not practical to manipulate the ice nucleation temperature as the cooling rates are limited inside the lyophilizer and the degree of supercooling might not change within such a small range.[1, 14] 3.2 Pre-cooled shelf method When applying the pre-cooled shelf method, the vials are placed on the lyophilizer shelf which is already cooled to the desired final shelf temperature, e.g. -40 °C or -45 °C.[1, 13-14] It is reported that the placement of samples on a pre-cooled shelf results in higher nucleation temperatures (-9,5 °C) compared to the conventional shelf-ramped freezing (-13.4 °C).[14] Moreover, with this lowered degree of supercooling and more limited time for thermal equilibration throughout the fill volume, the freezing rate after ice nucleation is actually slower compared to shelf-ramped freezing.[40]   In addition, a large heterogeneity in supercooling between vials is observed for this method.[14] A distinct influence of the loading shelf temperature on the nucleation temperature is described in literature.[13-14] Searles et al.[14] found that the nucleation temperatures for samples placed on a shelf at -44 °C were several degrees higher than for samples placed on a -40 °C shelf. Thus, when using this method the shelf temperature should be chosen with care. 3.3 Annealing Annealing is defined as a hold step at a temperature above the glass transition temperature.[12] In general, annealing is performed to allow for complete crystallization of crystalline compounds and to improve inter-vial heterogeneity and drying rates.[1, 19] Tang et al.[1] proposed the following annealing protocol: when the final shelf temperature is reached after the freezing step, the product temperature is increased to 10 to 20 °C above Tg` but well below Teu and held for several hours. Afterwards the shelf temperature is decreased to and held at the final shelf temperature. Annealing has a rigorous effect on the ice crystal size distribution [17, 41] and can delete the interdependence between the ice nucleation temperature and ice crystal size and morphology. If the sample temperature exceeds Tg`, the system pursues the equilibrium freezing curve and some of the ice melts.[12, 41] The raised water content and the increased temperature enhance the mobility of the amorphous phas e and all species in that phase.[12] This increased mobility of the amorphous phase enables the relaxation into physical states of lower free energy.[12] According to the Kelvin equation ice crystals with smaller radii of curvature will melt preferentially due to their higher free energy compared to larger ice crystals.[12, 37, 41] Ostwald ripening (recrystallization), which results in the growth of dispersed crystals larger than a critical size at the expense of smaller ones, is a consequence of these chemical potential driving forces.[12, 41] Upon refreezing of the annealed samples small ice crystals do not reform as the large ice crystals present serve as nucleation sites for addition crystallization.[41] The mean ice crystal radius rises with time1/3 during annealing.[37, 41] A consequence of that time dependency is that the inter-vial heterogeneity in ice crystal size distribution is reduced with increasing annealing time, as vials comprising smaller ice crystals â€Å"catch u p† with the vials that started annealing containing larger ice crystals.[12, 17, 37, 41] Searles et al.[41] found that due to annealing multiple sheets of lamellar ice crystals with a high surface area merged to form pseudo-cylindrical shapes with a lower interfacial area. In addition to the increase in ice crystal size, they observed that annealing opened up holes on the surface of the lyophilized cake. The hole formation is explained by the diffusion of water from melted ice crystals through the frozen matrix at the increased annealing temperature. Moreover, in the case of meta-stable glass formation of crystalline compounds, annealing facilitates complete crystallization.[42] Above Tg` the meta-stable glass is re-liquefied and crystallization occurs when enough time is provided. Furthermore, annealing can promote the completion of freeze concentration (devitrification) as it allows amorphous water to crystallize.[41] This is of importance when samples were frozen too fast a nd water capable of crystallization was entrapped as amorphous water in the glassy matrix. In addition, the phenomenon of annealing also becomes relevant when samples are optimal frozen but are then kept at suboptimal conditions in the lyophilizer or in a freezer before lyophilization is performed.[11] 3.4 Quench freezing During quench freezing, also referred to as vial immersion, the vials are immersed into either liquid nitrogen or liquid propane (ca. -200 °C) or a dry ice/ acetone or dry ice/ ethanol bath (ca. -80 °C) long enough for complete solidification and then placed on a pre-cooled shelf.[9, 16] In this case the heat-transfer media is in contact with both the vial bottom and the vial wall [10], leading to a ice crystal formation that starts at the vial wall and bottom. This freezing method results in a lowered degree of supercooling but also a high freezing rate as the sample temperature is decreased very fast, resulting in small ice crystals. Liquid nitrogen immersion has been described to induce less supercooling than slower methods [9, 37, 39] , but more precise this faster cooling method induces supercooling only in a small sample volume before nucleation starts and freezes by directional solidification.[12, 14]   While it is reported that external quench freezing might be advantag eous for some applications [39], this uncontrolled freezing method promotes heterogeneous ice crystal formation and is not applicable in large scale manufacturing.[7] 3.5 Directional freezing In order to generate straight, vertical ice crystallization, directional respectively vertical freezing can be performed. Here, ice nucleation is induced at the bottom of the vial by contact with dry ice and slow freezing on a pre-cooled shelf is followed.[9] In this case, the ice propagation is vertically and lamellar ice crystals are formed.[9] A similar approach, called unidirectional solidification, was described by Schoof et al. [43]. Here each sample was solidified in a gradient freezing stage, based on the Power-Down principle, with a temperature gradient between the upper and the lower cooling stage of 50 K/cm, resulting in homogenous ice-crystal morphology. 3.6 Ice-fog technique In 1990, Rowe [44] described an ice-fog technique for the controlled ice nucleation during freezing. After the vials are cooled on the lyophilizer shelf to the desired nucleation temperature, a flow of cold nitrogen is led into the chamber. The high humidity of the chamber generates an ice fog, a vapor suspension of small ice particles. The ice fog penetrates into the vials, where it initiates ice nucleation at the solutio Physico-chemical Processes that Occur During Freezing Physico-chemical Processes that Occur During Freezing 1. Introduction Lyophilization respectively freeze-drying is an important and well-established process to improve the long-term stability of labile drugs, especially therapeutic proteins.[1] About 50% of the currently marketed biopharmaceuticals are lyophilized, representing the most common formulation strategy.[2] In the freeze-dried solid state chemical or physical degradation reactions are inhibited or sufficiently decelerated, resulting in an improved long-term stability.[3] Besides the advantage of better stability, lyophilized formulations also provide easy handling during shipping and storage. [1] A traditional lyophilization cycle consists of three steps; freezing, primary drying and secondary drying.[1] During the freezing step, the liquid formulation is cooled until ice starts to nucleate, which is followed by ice growth, resulting in a separation of most of the water into ice crystals from a matrix of glassy and/or crystalline solutes.[4-5] During primary drying, the crystalline ice formed during freezing is removed by sublimation. Therefore, the chamber pressure is reduced well below the vapor pressure of ice and the shelf temperature is raised to supply the heat removed by ice sublimation.[6] At the completion of primary drying, the product can still contain approximately 15% to 20% of unfrozen water, which is desorbed during the secondary drying stage, usually at elevated temperature and low pressure, to finally achieve the desired low moisture content.[7] In general, lyophilization is a very time- and energy-intensive drying process.[8]   Typically, freezing is over within a few hours while drying often requires days. Within the drying phase, secondary drying is short (~hours) compared to primary drying (~days).[1, 4] Therefore, lyophilization cycle development has typically focused on optimizing the primary drying step, i.e., shortening the primary drying time by adjusting the shelf temperature and/or chamber pressure without influencing product quality.[5, 9] Although, freezing is one of the most critical stages during lyophilization, the importance of the freezing process has rather been neglected in the past.[10]   The freezing step is of paramount importance. At first, freezing itself is the major desiccation step in lyophilization [6] as solvent water is removed from the liquid formulation in the form of a pure solid ice phase, leading to a dramatic concentration of the solutes.[11-12] Moreover, the kinetics of ice nucleation and crystal growth determine the physical state and morphology of the frozen cake and consequently the final properties of the freeze-dried product.[11-13] Ice morphology is directly correlated with the rate of sublimation in primary and secondary drying.[14] In addition, freezing is a critical step with regard to the biological activity and stability of the active pharmaceutical ingredients (API), especially pharmaceutical proteins.[1] While simple in concept, the freezing process is presumably the most complex but also the most important step in the lyophilization process.[10] To meet this challenge, a thorough understanding of the physico-chemical processes, which occur during freezing, is required. Moreover, in order to optimize the freeze drying process and product quality, it is vital to control the freezing step, which is challenging because of the random nature of ice nucleation. However, several approaches have been developed to trigger ice nucleation during freezing. The purpose of this review is to provide the reader with an awareness of the importance but also complexity of the physico-chemical processes that occur during freezing. In addition, currently available freezing techniques are summarized and an attempt is made to address the consequences of the freezing procedure on process performance and product quality. A special focus is set on the critical factors that influence protein stability. Understanding and controlling the freezing step in lyophilization will lead to optimized, more efficient lyophilization cycles and products with an improved stability. 2. Physico-chemical fundamentals of freezing The freezing process first involves the cooling of the solution until ice nucleation occurs. Then ice crystals begin to grow at a certain rate, resulting in freeze concentration of the solution, a process that can result in both crystalline and amorphous solids, or in mixtures.[11] In general, freezing is defined as the process of ice crystallization from supercooled water.[15] The following section summarizes the physico-chemical fundamentals of freezing. At first, the distinction between cooling rate and freezing rate should be emphasized. The cooling rate is defined as the rate at which a solution is cooled, whereas the freezing rate is referred to as the rate of postnucleation ice crystal growth, which is largely determined by the amount of supercooling prior to nucleation.[16-17] Thus, the freezing rate of a formulation is not necessarily related to its cooling rate.[18] 2.1 Freezing phenomena: supercooling, ice nucleation and ice crystal formation In order to review the physico-chemical processes that occur during freezing of pure water, the relationship between time and temperature during freezing is displayed in figure 1. When pure water is cooled at atmospheric pressure, it does not freeze spontaneously at its equilibrium freezing point (0 °C).[19] This retention of the liquid state below the equilibrium freezing point of the solution is termed as â€Å"supercooling†.[19] Supercooling (represented by line A) always occurs during freezing and is often in the range of 10 to 15 °C or more.[12, 18] The degree of supercooling is defined as the difference between the equilibrium ice formation temperature and the actual temperature at which ice crystals first form and depends on the solution properties and process conditions.[1, 6, 11, 20] As discussed later, it is necessary to distinguish between â€Å"global supercooling†, in which the entire liquid volume exhibits a similar level of supercooling, and â€Å"lo cal supercooling†, in which only a small volume of the liquid is supercooled.[14] Supercooling is a non-equilibrium, meta-stable state, which is similar to an activation energy necessary for the nucleation process.[21] Due to density fluctuations from Brownian motion in the supercooled liquid water, water molecules form clusters with relatively long-living hydrogen bonds [22] almost with the same molecular arrangement as in ice crystals.[11, 15] As this process is energetically unfavorable, these clusters break up rapidly.[15] The probability for these nuclei to grow in both number and size is more pronounced at lowered temperature.[15] Once the critical mass of nuclei is reached, ice crystallization occurs rapidly in the entire system (point B).[15, 21-22]   The limiting nucleation temperature of water appears to be at about -40 °C, referred to as the â€Å"homogeneous nucleation temperature†, at which the pure water sample will contain at least one spontaneously f ormed active water nucleus, capable of initiating ice crystal growth.[11] However, in all pharmaceutical solutions and even in sterile-filtered water for injection, the nucleation observed is â€Å"heterogeneous nucleation†, meaning that ice-like clusters are formed via adsorption of layers of water on â€Å"foreign impurities†.[6, 11] Such â€Å"foreign impurities† may be the surface of the container, particulate contaminants present in the water, or even sites on large molecules such as proteins.[23-24] Primary nucleation is defined as the initial, heterogeneous ice nucleation event and it is rapidly followed by secondary nucleation, which moves with a front velocity on the order of mm/s through the solution. [14, 25] Often secondary nucleation is simply referred to as ice crystallization, and the front velocity is sometime referred to as the crystallization linear velocity.[14] Once stable ice crystals are formed, ice crystal growth proceeds by the addition of molecules to the interface.[22] However, only a fraction of the freezable water freezes immediately, as the supercooled water can absorb only 15cal/g of the 79cal/g of heat given off by the exothermic ice formation.[12, 22] Therefore, once crystallization begins, the product temperature rises rapidly to near the equilibrium freezing point.[12, 26] After the initial ice network has formed (point C), additional heat is removed from the solution by further cooling and the remaining water freezes when the previously formed ice crystals grow.[12] The ice crystal growth is controlled by the latent heat release and the cooling rate, to which the sample is exposed to.[22] The freezing time is defined as the time from the completed ice nucleation to the removal of latent heat (from point C to point D). The temperature drops when the freezing of the sample is completed (point E).[21] The number of ice nuclei formed, the rate of ice growth and thus the ice crystals` size depend on the degree of supercooling.[14, 20] The higher the degree of supercooling, the higher is the nucleation rate and the faster is the effective rate of freezing, resulting in a high number of small ice crystals. In contrast, at a low degree of supercooling, one observes a low number of large ice crystals.[14, 19] The rate of ice crystal growth can be expressed as a function of the degree of supercooling.[23]   For example for water for injection, showing a degree of supercooling of 10 °C +/- 3 °C, an ice crystal growth rate of about   5.2cm/s results.[23] In general, a slower cooling rate leads to a faster freezing rate and vice versa. Thus, in case of cooling rate versus freezing rate it has to be kept in mind â€Å"slow is fast and fast is slow†. Nevertheless, one has to distinguish between the two basic freezing mechanisms. When global supercooling occurs, which is typically the case for shelf-ramped freezing, the entire liquid volume achieves a similar level of supercooling and solidification progresses through the already nucleated volume.[12, 14] In contrast, directional solidification occurs when a small volume is supercooled, which is the case for high cooling rates, e.g. with nitrogen immersion. Here, the nucleation and solidification front are in close proximity in space and time and move further into non-nucleated solution. In this case, a faster cooling rate will lead to a faster freezing rate.[12, 14] Moreover, as ice nucleation is a stochastically event [6, 18], ice nucleation and in consequence ice crystal size distribution will differ from vial to vial resulting in a huge sample heterogeneity within one batch.[6, 14, 27] In addition, during freezing the growth of ice crystals within one vial can also be heterogeneous, influencing intra-vial uniformity.[5] Up to now, 10 polymorphic forms of ice are described. However, at temperatures and pressures typical for lyophilization, the stable crystal structure of ice is limited to the hexagonal type, in which each oxygen atom is tetrahedrally surrounded by four other oxygen atoms.[23] The fact that the ice crystal morphology is a unique function of the nucleation temperature was first reported by Tammann in 1925.[28] He found that frozen samples appeared dendritic at low supercoolings and like â€Å"crystal filaments† at high supercooling. In general, three different types of growth of ice crystals around nuclei can be observed in solution[15]: i) if the water molecules are given sufficient time, they arrange themselves regularly into hexagonal crystals, called dendrites; ii) if the water molecules are incorporated randomly into the crystal at a fast rate, â€Å"irregular dendrites† or axial columns that originate from the center of crystallization are formed; iii) at higher coo ling rates, many ice spears originate from the center of crystallization without side branches, referred to as spherulites. However, the ice morphology depends not only on the degree of supercooling but also on the freezing mechanism. It is reported that â€Å"global solidification† creates spherulitic ice crystals, whereas â€Å"directional solidification† results in directional lamellar morphologies with connected pores.[12, 14] While some solutes will have almost no effect on ice structure, other solutes can affect not only the ice structure but also its physical properties.[19] Especially at high concentrations, the presence of solutes will result in a depression of the freezing point of the solution based on Raoults`s Law and in a faster ice nucleation because of the promotion of heterogeneous nucleation, leading to a enormously lowered degree of supercooling.[21] 2.2 Crystallization and vitrification of solutes The hexagonal structure of ice is of paramount importance in lyophilization of pharmaceutical formulations, because most solutes cannot fit in the dense structure of the hexagonal ice, when ice forms.[23] Consequently, the concentration of the solute constituents of the formulation is increased in the interstitial region between the growing ice crystals, which is referred to as â€Å"cryoconcentration†.[11-12] If this separation would not take place, a solid solution would be formed, with a greatly reduced vapor pressure and the formulation cannot be lyophilized.[23] The total solute concentration increases rapidly and is only a function of the temperature and independent of the initial concentration.[4] For example, for an isotonic saline solution a 20-fold concentration increase is reported when cooled to -10 °C and all other components in a mixture will show similar concentration increases.[4] Upon further cooling the solution will increase to a critical concentration, ab ove which the concentrated solution will either undergo eutectic freezing or vitrification.[7] A simple behavior is crystallization of solutes from cryoconcentrated solution to form an eutectic mixture.[19] For example, mannitol, glycine, sodium chloride and phosphate buffers are known to crystallize upon freezing, if present as the major component.[12] When such a solution is cooled, pure ice crystals will form first. Two phases are present, ice and freeze-concentrated solution. The composition is determined via the equilibrium freezing curve of water in the presence of the solute (figure 2). The system will then follow the specific equilibrium freezing curve, as the solute content increases because more pure water is removed via ice formation. At a certain temperature, the eutectic melting temperature (Teu), and at a certain solute concentration (Ceu), the freezing curve will meet the solubility curve. Here, the freeze concentrate is saturated and eutectic freezing, which means solute crystallization, will occur.[7, 19] Only below Teu, which is defined as the lowest temperat ure at which the solute remains a liquid the system is completely solidified.[19] The Teu and Ceu are independent of the initial concentration of the solution.[7] In general, the lower the solubility of a given solute in water, the higher is the Teu.[19] For multicomponent systems, a general rule is that the crystallization of any component is influenced, i.e. retarded, by other components.[11] In practice, analogous to the supercooling of water, only a few solutes will spontaneously crystallize at Teu.[11] Such delayed crystallization of solutes from a freezing solution is termed supersaturation and can lead to an even more extreme freeze concentration.[11] Moreover, supersaturation can inhibit complete crystallization leading to a meta-stable glass formation, e.g. of mannitol.[12, 23] In addition, it is also possible that crystalline states exist in a mixture of different polymorphs or as hydrates.[11] For example, mannitol can exist in the form of several polymorphs (a, b and d) und under certain processing conditions, it can crystallize as a monohydrate.[11] The phase behavior is totally different for polyhydroxy compounds like sucrose, which do not crystallize at all from a freezing solution in real time.[11] The fact that sucrose does not crystallize during freeze-concentration is an indication of its extremely complex crystal structure.[11] The interactions between sugar -OH groups and those between sugar -OH groups and water molecules are closely similar in energy and configuration, resulting in very low nucleation probabilities.[11] In this case, water continues to freeze beyond the eutectic melting temperature and the solution becomes increasingly supersaturated and viscous.[11] The increasing viscosity slows down ice crystallization, until at some characteristic temperature no further freezing occurs.[11] This is called glassification or vitrification.[18]   The temperature at which the maximal freeze-concentration (Cg`) occurs is referred to as the glass transition temperature Tg`.[11, 29] This point is at the intersection of t he freezing point depression curve and the glass transition or isoviscosity curve, described in the â€Å"supplemented phase diagram† [30] or â€Å"state diagram† (figure 2).[11] Tg ´ is the point on the glass transition curve, representing a reversible change between viscous, rubber-like liquid and rigid, glass system.[19] In the region of the glass transition, the viscosity of the freeze concentrate changes about four orders of magnitude over a temperature range of a few degrees.[19] Tg` depends on the composition of the solution, but is independent of the initial concentration.[4, 11, 27]   For example, for the maximally freeze concentration of sucrose a concentration of 72-73% is reported.[31] In addition to Tg` the collapse temperature (Tc) of a product is used to define more precisely the temperature at which a structural loss of the product will occur. In general Tc is several degrees higher than Tg`, as the high viscosity of the sample close to Tg` will pre vent .[10] The glassy state is a solid solution of concentrated solutes and unfrozen, amorphous water. It is thermodynamically unstable with respect to the crystal form, but the viscosity is high enough, in the order of 1014 Pa*s, that any motion is in the order of mm/year.[4, 11, 29] The important difference between eutectic crystallization and vitrification is that for crystalline material, the interstitial between the ice crystal matrix consists of an intimate mixture of small crystals of ice and solute, whereas for amorphous solutes, the interstitial region consists of solid solution and unfrozen, amorphous water.[19, 23] Thus, for crystalline material nearly all water is frozen and can easily be removed during primary drying without requiring secondary drying.[19] However, for amorphous solutes, about 20% of unfrozen water is associated in the solid solution, which must be removed by a diffusion process during secondary drying.[19] Moreover, the Teu for crystalline material or the Tg` respectively Tc for amorphous material define the maximal allowable product temperature during primary drying.[19] Eutectic melting temperatures are relatively high compared to glass transition temperatures, allowing a higher product temperature during primary drying, which resu lts in more efficient drying processes.[19] If the product temperature exceeds this critical temperature crystalline melting or amorphous collapse will occur, resulting in a loss of structure in the freeze-dried product, which is termed â€Å"cake collapse†.[11, 19] 2.3 Phase separation and other types of freezing behavior A characteristic property of multicomponent aqueous solutions, especially when at least one component is a polymer, is the occurrence of a liquid-liquid phase separation during freezing into two liquid equilibrium phases, which are enriched in one component.[11, 19] This phase separation behavior has been reported for aqueous solutions of polymers such as PEG/dextran or PVP/dextran but is also reported for proteins and excipients.[32-33] When a critical concentration of the solutes is reached, the enthalpically unfavorable interactions between the solutes exceed the favorable entropy of a solution with complete miscibility.[34] Another proposed explanation is that solutes have different effects on the structure of water, leading to phase separation.[35] Besides the separation into two amorphous phases, two other types of phase separation are stated in literature; crystallization of amorphous solids and amorphization from crystalline solids.[18] Crystallization of amorphous solids often occurs when metastable glasses are formed during freezing. In this case, e.g. upon extremely fast cooling, a compound that normally would crystallize during slower freezing is entrapped as an amorphous, metastable glass in the freeze-concentrate.[12, 23] However, with subsequent heating above Tg`, it will undergo crystallization, which is the basis for annealing during freeze-drying (see 3.3).[19] Without annealing, the metastable glass can crystallize spontaneously out of the amorphous phase during drying or storage.[18] Amorphization from crystalline solids, that can be buffer components or stabilizers, predominantly occurs during the drying step and not during the freezing step.[18, 36]   Additionally, lyotropic liquid crystals, which have the degree of order between amorphous and crystalline, are reported to form as a result of freeze-concentration. However, their influence on critical quality attributes of the lyophilized product are not clarified.[19] Moreover, clathrates, also termed gas hydrates, are known to form, especially in the presence of non-aqueous co-solvents, when the solute alters the structure of the water.[23] 3. Modifications of the freezing step As aforementioned, the ice nucleation temperature defines the size, number and morphology of the ice crystals formed during freezing. Therefore, the statistical nature of ice nucleation poses a major challenge for process control during lyophilization. This highlights the importance of a controlled, reproducible and homogeneous freezing process. Several methods have been developed in order to control and optimize the freezing step. Some of them only intend to influence ice nucleation by modifying the cooling rate. Others just statistically increase the mean nucleation temperature, while a few allow a true control of the nucleation at the desired nucleation temperature. 3.1 Shelf-ramped freezing Shelf-ramped freezing is the most often employed, conventional freezing condition in lyophilization.[37] Here, at first, the filled vials are placed on the shelves of the lyophilizer and the shelf temperature is then decreased linearly (0.1 °C/min up to 5 °C/min, depending on the capacity of the lyophilizer) with time.[37-38] As both water and ice have low thermal conductivities and large heat capacities and as the thermal conductivity between vials and shelf is limited, the shelf-ramped cooling rate is by nature slow.[11] In order to ensure the complete solidification of the samples, the samples must be cooled below Tg` for amorphous material respectively below Teu for crystalline material. Traditionally, many lyophilization cycles use a final shelf temperature of -50 °C or lower, as this was the maximal cooling temperature of the freeze-drier.[7] Nowadays, it is suggested to use a final shelf temperature of -40 °C if the Tg` or Teu is higher than -38 °C or to use a temper ature of 2 °C less than Tg` and Teu.[1] Moreover, complete solidification requires significant time.[11] In general, the time for complete solidification depends on the fill volume; the larger the fill volume the more time is required for complete solidification.[11] Tang et al.[1]   suggest that the final shelf temperature should be held for 1 h for samples with a fill depth of less than or equal to 1 cm or 2 h for samples with a fill depth of greater than 1 cm. Moreover, fill depth of greater than 2 cm should be avoided, but if required, the holding time should be increased proportionately. In order to obtain a more homogeneous freezing, often the vials are equilibrated for about 15 to 30 min at a lowered shelf temperature (5 °C 10 °C) before the shelf temperature is linearly decreased.[1] Here, either the vials are directly loaded on the cooled shelves or the vials are loaded at ambient temperature and the shelf temperature is decreased to the hold temperature. [1, 5, 9] Another modification of the shelf-ramped freezing is the two-step freezing, where a â€Å"supercooling holding† is applied.(7) Here, the shelf temperature is decreased from room temperature or from a preset lowered shelf temperature to about -5 to -10 °C for 30 to 60min hold. This leads to a more homogenous supercooling state across the total fill volume.[1, 5] When the shelf temperature is then further decreased, relatively homogeneous ice formation is observed.[5] In general, shelf-ramped frozen samples show a high degree of supercooling but when the nucleation temperature is reached, ice crystal growth proceeds extremely fast, resulting in many small ice crystals.[9, 39] However, the ice nucleation cannot be directly controlled when shelf-ramped freezing is applied and is therefore quite random.[4] Thus, one drawback of shelf-ramped freezing is that different vials may become subject to different degrees of supercooling, typically about +/- 3 °C about the mean.[4] This results in a great variability in product quality and process performance.[4] Moreover, with the shelf-ramped freezing method it is not practical to manipulate the ice nucleation temperature as the cooling rates are limited inside the lyophilizer and the degree of supercooling might not change within such a small range.[1, 14] 3.2 Pre-cooled shelf method When applying the pre-cooled shelf method, the vials are placed on the lyophilizer shelf which is already cooled to the desired final shelf temperature, e.g. -40 °C or -45 °C.[1, 13-14] It is reported that the placement of samples on a pre-cooled shelf results in higher nucleation temperatures (-9,5 °C) compared to the conventional shelf-ramped freezing (-13.4 °C).[14] Moreover, with this lowered degree of supercooling and more limited time for thermal equilibration throughout the fill volume, the freezing rate after ice nucleation is actually slower compared to shelf-ramped freezing.[40]   In addition, a large heterogeneity in supercooling between vials is observed for this method.[14] A distinct influence of the loading shelf temperature on the nucleation temperature is described in literature.[13-14] Searles et al.[14] found that the nucleation temperatures for samples placed on a shelf at -44 °C were several degrees higher than for samples placed on a -40 °C shelf. Thus, when using this method the shelf temperature should be chosen with care. 3.3 Annealing Annealing is defined as a hold step at a temperature above the glass transition temperature.[12] In general, annealing is performed to allow for complete crystallization of crystalline compounds and to improve inter-vial heterogeneity and drying rates.[1, 19] Tang et al.[1] proposed the following annealing protocol: when the final shelf temperature is reached after the freezing step, the product temperature is increased to 10 to 20 °C above Tg` but well below Teu and held for several hours. Afterwards the shelf temperature is decreased to and held at the final shelf temperature. Annealing has a rigorous effect on the ice crystal size distribution [17, 41] and can delete the interdependence between the ice nucleation temperature and ice crystal size and morphology. If the sample temperature exceeds Tg`, the system pursues the equilibrium freezing curve and some of the ice melts.[12, 41] The raised water content and the increased temperature enhance the mobility of the amorphous phas e and all species in that phase.[12] This increased mobility of the amorphous phase enables the relaxation into physical states of lower free energy.[12] According to the Kelvin equation ice crystals with smaller radii of curvature will melt preferentially due to their higher free energy compared to larger ice crystals.[12, 37, 41] Ostwald ripening (recrystallization), which results in the growth of dispersed crystals larger than a critical size at the expense of smaller ones, is a consequence of these chemical potential driving forces.[12, 41] Upon refreezing of the annealed samples small ice crystals do not reform as the large ice crystals present serve as nucleation sites for addition crystallization.[41] The mean ice crystal radius rises with time1/3 during annealing.[37, 41] A consequence of that time dependency is that the inter-vial heterogeneity in ice crystal size distribution is reduced with increasing annealing time, as vials comprising smaller ice crystals â€Å"catch u p† with the vials that started annealing containing larger ice crystals.[12, 17, 37, 41] Searles et al.[41] found that due to annealing multiple sheets of lamellar ice crystals with a high surface area merged to form pseudo-cylindrical shapes with a lower interfacial area. In addition to the increase in ice crystal size, they observed that annealing opened up holes on the surface of the lyophilized cake. The hole formation is explained by the diffusion of water from melted ice crystals through the frozen matrix at the increased annealing temperature. Moreover, in the case of meta-stable glass formation of crystalline compounds, annealing facilitates complete crystallization.[42] Above Tg` the meta-stable glass is re-liquefied and crystallization occurs when enough time is provided. Furthermore, annealing can promote the completion of freeze concentration (devitrification) as it allows amorphous water to crystallize.[41] This is of importance when samples were frozen too fast a nd water capable of crystallization was entrapped as amorphous water in the glassy matrix. In addition, the phenomenon of annealing also becomes relevant when samples are optimal frozen but are then kept at suboptimal conditions in the lyophilizer or in a freezer before lyophilization is performed.[11] 3.4 Quench freezing During quench freezing, also referred to as vial immersion, the vials are immersed into either liquid nitrogen or liquid propane (ca. -200 °C) or a dry ice/ acetone or dry ice/ ethanol bath (ca. -80 °C) long enough for complete solidification and then placed on a pre-cooled shelf.[9, 16] In this case the heat-transfer media is in contact with both the vial bottom and the vial wall [10], leading to a ice crystal formation that starts at the vial wall and bottom. This freezing method results in a lowered degree of supercooling but also a high freezing rate as the sample temperature is decreased very fast, resulting in small ice crystals. Liquid nitrogen immersion has been described to induce less supercooling than slower methods [9, 37, 39] , but more precise this faster cooling method induces supercooling only in a small sample volume before nucleation starts and freezes by directional solidification.[12, 14]   While it is reported that external quench freezing might be advantag eous for some applications [39], this uncontrolled freezing method promotes heterogeneous ice crystal formation and is not applicable in large scale manufacturing.[7] 3.5 Directional freezing In order to generate straight, vertical ice crystallization, directional respectively vertical freezing can be performed. Here, ice nucleation is induced at the bottom of the vial by contact with dry ice and slow freezing on a pre-cooled shelf is followed.[9] In this case, the ice propagation is vertically and lamellar ice crystals are formed.[9] A similar approach, called unidirectional solidification, was described by Schoof et al. [43]. Here each sample was solidified in a gradient freezing stage, based on the Power-Down principle, with a temperature gradient between the upper and the lower cooling stage of 50 K/cm, resulting in homogenous ice-crystal morphology. 3.6 Ice-fog technique In 1990, Rowe [44] described an ice-fog technique for the controlled ice nucleation during freezing. After the vials are cooled on the lyophilizer shelf to the desired nucleation temperature, a flow of cold nitrogen is led into the chamber. The high humidity of the chamber generates an ice fog, a vapor suspension of small ice particles. The ice fog penetrates into the vials, where it initiates ice nucleation at the solutio

The Nature of Construction Problems: Past and Present

The Nature of Construction Problems: Past and Present ABSTRACT: This paper aims at deriving a comprehensive understanding pertaining to the different construction difficulties that have been faced in the past and are being faced now in the present also. It provides a vivid analysis of why the problems were faced, the nature of the problems and how they were dealt with. It has been a long time since the first inception of architectural ideas that had crept into our ancestors’ minds. Right from the Stone Age, man has actually been into this act of constructing stuff for making his life easy. At the very juncture of the Metal Age, he got a further boost with the advent of new materials and construction techniques. Furthermore, came the times when architecture was at its peak. Everywhere in the world there was zeal to construct structures that not only made life comfortable for man, but also soothed his eyes. The classical orders of the Greeks packed the punch of serving as better structural members along with being a visual treat. The Romans had been articulate in constructing an efficient system of water supply in the form of a series of arches together being called as aquaducts. It still stands as an example people look up to design urban spaces. Egyptians were no less. They were meticulously constructing pyramids and mortuary tombs that were way beyond the human scale. They had designed the first forms of conveyor systems in building construction. Similarly, India saw the construction of a lot of architectural wonders. Right from the construction of the monolithic stone structures of the Ajanta-Ellora caves and the temples of Mahabalipuram to the Islamic structures of Taj Mahal and the Humayun’s tomb it had been a marvellous leap in the form of architectural progress. Finally came the times of controversies about architecture when Gustav Eiffel proposed the construction of an all metal structure now called the Eiffel tower. There was a lot of noise about the fact that how it would finally look when it was constructed in Paris. Many people said that it might turn out to be a scar on the face of the city. But, now fortunately it is known as the first thing that helps us identify the city of Paris. There have also been controversies about Zaha Hadid’s designs. She is one of the pioneer architects as far as t he use of fluidic forms is concerned. Her designs have almost always been at the helm of criticism by the senior architects of her times. But, she has been articulate in executing her own designs. Similarly, in the recent times Frank O’ Gehry had faced a lot of challenges in bringing up his work called Bilbao museum. He has been criticised like hell up to the level of being called the mad architect. Now as we come up to the fact of such progress in the field of construction, can we deny that there have been no difficulties in the construction of these age old structures? No will be the answer. As we go back to the times when the monolithic stone structures of the Kailasa temple was constructed, we get to know that no good technology was available at that time. However, they have managed to actually make it possible to form a massive temple out of a single rock. Imagine that how the person who started the construction, actually just used a chisel and a hammer to build such a huge temple. It is said that he started from the top and kept on chiselling till the bottom. It is evident that this wonderful construction must have taken a long time to come up. But, it has not been constructed by a single sculptor. Imagine the difficulty in passing on the same knowledge about the structure to so many artisans over the ages. There might have been several changes made to this temple structure during the construction. This must have made it even more difficult for the sculptors. Now raises the question of managing the waste that was generated in this process. How were all the cut out st ones used? Yes, they were used to make roads and for pety constructions. But wasn’t it really difficult to manage these heavy masses? Yes, it must have been tedious. A similar structure had come up previously. The Konark temple in Bhubaneshwar, Orissa had a huge contribution in raising the architectural taste of the people of the country. In this construction, they used the method of constructing sand ramps as it was being constructed towards the top end. This had actually ensured that the structure didn’t get decayed during the construction process. However, this structure had made use of a huge pole at the centre in the form of a magnet that gave the massive stone structure all its strength. This had been acquired by the British during their reign in our country. But, later on what it was used for nobody knows. Just imagine how that huge magnet was made to stand just by the use of very basic tools. This speaks of the difficulty in constructing structures belonging to the typology of stone temples. Now, when the importance of Constantinople came into existence, there was a sudden need for the construction of several public buildings. At that time there was a huge recession in the field of construction in Europe. There was a shortage of resources, funds, architects and even labourers. This thing gave rise to a new form of architecture. This form of architecture came to be known as Polymath Architecture. In this form of architecture the existing buildings were used to build several other buildings. The Roman basilicas were used to build the Basilican churches that formed the basis of Christianity. The columns of the condemned buildings were actually truncated to get a uniform size. As a result, the buildings were having a size very close to the human scale. Explicitly or purposely, this form of architecture happened to be more close to people. Even the foundations of several buildings were used to meet the needs of the times. The rotunda buildings were used to used to build bapti steries when the question of child baptism came into existence. These spaces required a kind of radiating building plan in order to accommodate the required activity. So, the unavailability of resources at that time was addressed in such a way. Now, when we come to construction of buildings that are built with the on public money, the question of minimalism comes into existence. The usage of minimum amount of resource and funds comes into existence. Taking the example of a case, when an architect is given task of designing a public building, he has to make sure that he does not raise the cost of the building to the height of making it unfeasible. Nowadays, in case of construction of public buildings, the factor of tender pops up. The person coming up with the most apt design within the least of cost frame usually gets the project. Such a case can be taken up when it comes to the Bilbao museum. It was designed by Frank O’ Gehry. He had gone through the criticism of the entire world because of his design. His design was so complex that it made use of aircraft design software. He had actually gone to the point of overusing resources. The frames of the building were made using thick steel sections. A large layer of fabri c was used to cover the building. Above this a layer of titanium sheets were used. Around twenty four thousand square meters of such were used. As a result of this, he was criticised over the fact that a similar functionality and aesthetics was achievable with lesser use of resources. But, he had paid no heed to what the people said. He has a concept of a fish just because he had memories of going to a fish market with his grandma. This vague justification brought about a lot of talks throughout the world. Luckily, the titanium prices had gone down at that point of time. So, he had the privilege of not getting the project cancelled. When residential projects are taken into consideration, there are several pety issues that come up. Funds remain limited in most cases. There have been cases when construction of a single house has gone through a ceasework period of more than a year just due to the lack of funds. Other religious issues play a vital role in our country when it comes to construction of residences specially. Several people actually back out when the architect is reluctant to listen to his specifications about how the building should be. Industrial buildings have challenges that are totally different as compared to other buildings. They should have the ability to actually justify all the government norms regarding the minute points like waste generation and hazard safety factors. They should have special anthropometric specifications but they should not use too much of space. So, as we come to conclude this, we can actually see that several issues are getting solved in this context. Several architects are coming up with innovative ideas to actually cater to the ideas of the government and the environmental impact assessment bodies to make a statement regarding this matter. So let us hope for a hassle free and green future ahead!!! References: History Of Architecture by Sir Bannister Fletcher Construction Delays: Extension Of time and prolongation claims by Roger Gibson

Progressivism :: Teaching Education Essays

Progressivism Progressivists believe that people learn best from what they consider most relevant to their lives. Therefore, the curriculum should be centered around the experiences, interests, and abilities of students. Teachers should plan lessons that arouse curiosity and push the students to a higher level of knowledge. In addition to reading textbooks, the students must learn by doing. Students should often leave the classroom for fieldtrips during which they interact with nature or society. Teachers should also stimulate the students' interests through thought-provoking games. Students should be encouraged to interact with one another and to develop social virtues such as cooperation and tolerance for different points of view. Also, teachers should feel no compulsion to focus their students' attentions on one discrete discipline at a time, and students may be responsible for learning lessons that combine several different subjects. Progressivists emphasize in their curriculum the study of the natural and social sciences. They believe that teachers should expose students to many new scientific, technological, and social developments, reflecting their notion that progress and change are fundamental. Students should also be exposed to a more democratic curriculum that recognizes accomplishments of women and minorities as well as white males. In addition, students should solve problems in the classroom similar to those they will encounter outside of the schoolhouse; they will learn to be flexible problem solvers. Progressivists believe that education should be a perpetually enriching process of ongoing growth, not merely a preparation for adult lives. They also deny the essentialist belief that the study of traditional subject matter is appropriate for all students, regardless of interest and personal experience.

Japanese New Year Essay -- Japan History Holidays Essays

Japanese New Year Introduction: In Japan one of the most celebrated festivals is New Years. The Japanese New Year, or Oshogatsu (‚ ¨Ã‚ ³Ã…’Å ½), is given more preparation than any other festival. There are many traditions and activities that happen at this time. Oshogatsu is a time to visit temples, forget the troubles of the past year, and wish for good fortune in the new. Many festivals around this time also reflect the values and ideals of New Years. History: During the time of the Empress Suiko (593-628), Japan was influenced greatly by the religion and beliefs of China. In A.D. 604 Japan officially adopted the Chinese calendar. gThe new calendar was a lunar- solar one that has its months based on the lunar cycle. Casal writes gThe month begins when the sun enters the sign of the Fishes: the first day of the year, therefore falls on a day between January 20th and February 19th, and this is much nearer to spring (2). This system was in effect until the introduction of the Gregorian calendar in 1872. This is important because the seasons fall in line better with the lunar calendar than the modern one. In rural areas, many farmers still apply the Chinese calendar to their trade. Japanese people believed that the New Year started when the plants began to sprout life again, a new beginning. This happens throughout most of Japan in mid Jan. in line with the lunar calendar. In todayfs society Oshogatsu always falls on the same day (Jan. 1st), there are other festivals, which fall into the lunar calendar that have the same values of purification and new beginnings. Another influence from China at this time was Buddhism, which had been praised by Prince Shotoku Taishi, and became the official religion in 621.... ...nt from, and happier than, we do on ordinary days.h(Casal 1) Bibliography Festivals.com. 28 Nov. 2003 Seattle, Washington, http://www.festivals.com/search/display_event.aspx?eid=mYJmQbyD5jQ%3d&srid=3&page=1&bycountry=1&RID=jp&PROV= http://www.festivals.com/search/display_event.aspx?eid=lvluK1qWwM8%3d&srid=7&page=1&bycountry=1&RID=jp&PROV= (7) Japan Cultural Network Homepage. 28 Nov. 2003 http://www.hevanet.com/miyumi/december.htm http://www.hevanet.com/miyumi/january.htm http://www.hevanet.com/miyumi/february.htm Japan-Guide.com 2 Dec. 2003 Casal, U. A. The Five Sacred Festivals of Ancient Japan. Ruttland, Vermont &Tokyo Japan: Charles E. Tuttle Company Inc. 1967 Bauer, Helen & Carlquist, Sherwin. Japanese Festivals Doubleday & Company Inc. Garden City, New York, 1965 gHadaka Mairih Akita sakigake (Newspaper) 20 Jan. 2003 (1, 26)

Surrogate Motherhood Essay -- essays research papers fc

Surrogate Motherhood: Good or Bad?   Ã‚  Ã‚  Ã‚  Ã‚  There are many controversies surrounding the idea of surrogate motherhood, by its definition, it is a course of action that goes outside natural reproduction. Although surrogacy was first brought up in the bible it is only until recently that it has actually become an issue for criticism and debate. Factors such as the growth of infertility in modern society, coupled with the declining number of children available for adoption, and the development of surrogacy contract and commercial surrogacy agencies in 1976, have resulted in increasing publicity and public interest in the formation of agreements between infertile couples and surrogate mothers (Stuhmcke, 3). Surrogate motherhood can be approached in more than one way, and some surrogacy procedures receive more condemnation than others.   Ã‚  Ã‚  Ã‚  Ã‚  One type of surrogacy is when an egg is extracted from the infertile female and placed in a petri dish and is then fertilized by the fertile male’s sperm, the fertilized egg is then placed into the fertile female’s uterus so she can give birth to the child. This procedure is done when the uterus of a female is in some way defective, but her eggs are in fine condition, this is called invitro fertilization surrogacy or IVF surrogacy (Hanafin, 3). IVF surrogacy, or total surrogacy is the procedure that receives the least criticism because the baby is in no way related to its carrier. The more comm...

Fiat Pushes Work Ethic at Italian Plant

Flat which accounts for the evildoer of about 15000 families In the PANGOLIN D'ART region has Its least productive plant amongst all there. This Is due to the fact of employees misusing work ethics by being absent from work, faking illness and a doctor's note for as little as a local team's soccer match. FIAT suffered tremendous losses few years prior due to reduced production and quality issues. To counter this Fiat hired Sergei Maraschino as CEO who was expected to deliver similar success under his leadership as he did for American automaker Chrysler, rescuing the brand from bankruptcy.His motivation was to safeguard Jobs by pushing workers to be more devoted in turn boosting production and which would mirror Italy's economic standpoint and GAP. HIS vision for the need of change and pushing the Italians towards similar economic security like the Scandinavians was not accepted well. Nell Angelo a factory worker stated how Serious American methods were no good In Italy, where people for generations have lived with a slow paced lifestyle Insuring flexibility for workers.He also criticized Sergei for pulling the leash on absence and fearing a similar fate as Chinese workers who committed suicide due to excessive Rockford and stress. His radical solutions forced workers to change their routines and work ethics on the promise of bringing back production of the Panda back from the Polish people, creating more Jobs and working hours. Serious idea of changing working habits in order to improve individual financial futures and as a nation lying on the verge of sinking into debt's betterment signified his approach of the utilitarian normative theory.Under this theory Serious actions on misconduct and misuse of work ethics was beneficial for him and everyone in his surroundings. His actions and sessions focused on common welfare of not only employees but also Italy as a whole. Sergei before his utilitarian approach studied all possible factors which made the problem: Ser gei concluded individual factors such as cultural and social behavior and situational factors that influenced the psyche of employees since early adulthood that lead to the ethically incorrect behavior of employees on the global market sphere.He recognized how the Italian culture was different from his American experience/background and how it was normal for Italians to skip work and misuse power. The article also made clear how employees lacked Ethics of duty towards their workplace. 2. Determination of the dilemma: Sergei after his research about the Italian work atmosphere studied the extent of damage the unethical employee behavior could have had on them, FIAT and Italy as a nation. In his mind the bigger picture displayed of Italy ending debt-ridden was severe.He as a leader took responsibility for making a turnaround and saving Jobs which were livelihood for families residing in poor areas with increasing unemployment. 3. Course of action: Sergei took note to his previous vent ures and brainstormed with other veteran FIAT employees who shared similar mindset. He also consulted reports by economists Divide Strop on how if FIAT went down, Italy would too. He studied on how he could motivate workers to increase production. 4.Considering consequences: Sergei from the beginning knew his vision and opinion would not be accepted by Italian workers. They considered him an outsider whose American-style standards would force people to commit suicides. This was the biggest hurdle for Sergei for his utilitarian approach which by many was Judged egoistic and only good for him and FIAT. 5. Implementing the solution: Sergei implemented strict measures which were stated to be FIAT's curtain drawing on a humane working life and social upbringing of the Italians.His decisions cut absenteeism from 30% to 3% within the whole FIAT brand. His rule of sending doctors to homes of sick employees and workers for genuineness and penalizing by smaller lunch breaks did the trick. 6. Analyzing results : Serried measures proved to be successful as by the end of the year 63% of the employees signed FIAT's new working ethic model and preferred keeping a Job than being unemployed.

Albert Einstein: One Genius Mind

Finding a way to describe one of the most celebrated scientists ever born by means of stating facts about his life and achievements which most people hardly ever know at this point in time is utterly hard.   Albert Einstein’s father, an engineer by profession was for the most part of his career hugely unsuccessful.The older Einstein spent most of his time shifting jobs (Pellegrini 1).   Albert never enjoyed his education in Germany.He was seriously depressed when he had to leave for Italy.   He left school on the excuse of a nervous breakdown.   For a year, he had been paying visits to different art centers in Italy until such time when although reluctant, he finally resumed his studies.   It was in Zurich’s Eidgenossische Technische Hochschule (ETH) technical university where Einstein earned his degree (Pellegrini 1).He received poor grades since he hardly attended class lectures.   His poor credentials made it hard for him to secure a job.   Finally, a f riend helped him secure a patent office job in Berne (Pellegrini 1; AllAboutScience.org).It seemed that the patent job suits him perfectly.   It allowed him to work during the day and devote the rest of his time exploring higher learning in physics (White 96).   Also, he was able to utilize the library to do his research work.   He even earned for himself a doctorate degree in the processEven though it was not entirely all plain sailing from then on, several years later, he assumed a role in the academic system, and worked in Berlin to complete his General Theory of Relativity in the face of the severe destruction in society brought about by the First World War (Pellegrini 1).A serious breakdown left the man with distinct strands of white hair which ultimately served as his trademark.   It was around that time when his married life took a turn for the worst.   Nonetheless, he was still able to make significant contributions to scientific advancement.He was primarily, the e ngineer of breakthrough concepts about the physical universe.   Beyond doubt, he has made significant contributions to humanity’s intellectual advancement during his time.   Einstein challenged the then existing beliefs about the state as well as that of science.   He engaged in a constant battle for individual freedom and dignity.   He also opposed hostilities and persecution.His theories about the state and science, or at least the ones credited to him, echoes up to this point in time in realms stretching as far as his own direct scientific contributions in examining the influences that science and technology has on ethics, the study of modern art, linguistics, and psychology (Trefil and Hazen; White 96-105).It is paradoxical that several years following his death, several branches of physical science have already acknowledged his generative impact as otherwise would gave been done in the last years of his life.   Einstein’s contributions proved to be cruci al for drafting conceptual routes for current studies in the fields of cosmology or astronomy, for connecting gravitation with the quantum field theory of gauge fields, and also in explaining new findings which were not possible during his time, but were nonetheless already forecasted by him.Albert Einstein has the mind of a genius.   The way he changed the way man perceived the universe into which he or she was born was beyond comparison.   He was able to look past nature’s drape than anyone else since Newton, and from that point onwards, he lived the rest of his life pulling the drape down for his humility (Pellegrini 1).   To this day, when the word genius is being uttered, no one else’s face come to mind than his.