RESUMO
Best practices for performing freeze dryer equipment qualification are recommended, focusing on identifying methods to quantify shelf thermal uniformity (also known as "shelf surface uniformity"), equipment capability, and performance metrics of the freeze dryer essential to the pharmaceutical Quality by Design paradigm. Specific guidelines for performing shelf temperature mapping, freeze dryer equipment limit testing (the capability curve), and condenser performance metrics have been provided. Concerning shelf temperature mapping and equipment capability measurements, the importance of paying attention to the test setup and the use of appropriate testing tools are stressed. In all the guidelines provided, much attention has been paid to identifying the balance between obtaining useful process knowledge, logistical challenges associated with testing in the production environment vs that at laboratory scale, and the frequency of the testing necessary to obtain such useful information. Furthermore, merits and demerits of thermal conditions maintained on the cooled surfaces of the freeze dryer condenser have been discussed identifying the specific influence of the condenser surface temperature on the process conditions using experimental data to support the guidelines. Finally, guidelines for systematic leak rate testing criteria for a freeze dryer are presented. These specific procedural recommendations are based on calculations, measurements, and experience to provide useful process and equipment knowledge.
Assuntos
Liofilização , Tecnologia Farmacêutica , Liofilização/instrumentação , Tecnologia Farmacêutica/métodos , Temperatura , Guias como AssuntoRESUMO
PURPOSE: Hydrogen/deuterium (H/D) exchange over a range of temperatures suggests a protein structural/mobility transition in the solid state below the system glass transition temperature (Tg). The purpose of this study was to determine whether solid-state protein stability correlates with the difference between storage temperature and apparent Td where an abrupt change in mobility occurs, or alternatively, the extent of H/D exchange at a single temperature correlates directly to protein stability in lyophilized solids. METHODS: Solid-state H/D exchange was monitored by FTIR spectroscopy to study the extent of exchange and the apparent transition temperature in both pure recombinant human serum albumin (rHSA) and rHSA formulated with sucrose or trehalose. H/D exchange of freeze-dried formulations at 11% RH and temperatures from 30 to 80°C was monitored. Protein stability against aggregation at 40°C/11% RH for 6 months was assessed by size exclusion chromatography (SEC). RESULTS: Both sucrose and trehalose showed equivalent protection of protein secondary structure by FTIR. The rHSA:sucrose formulation showed superior long-term stability at 40°C by SEC over the trehalose formulation, but the apparent Td determined from H/D exchange was much higher in the trehalose formulation. Instead, the extent of H/D exchange (X∞) was lower in the sucrose formulation at the temperature of the stability studies (40°C) than found for the trehalose formulation, which was consistent with better stability in the sucrose formulation. CONCLUSIONS: While apparent Td did not correlate with protein stability for rHSA, the extent of H/D exchange, X∞, did.
Assuntos
Estabilidade Proteica , Proteínas Recombinantes/química , Albumina Sérica Humana/química , Físico-Química , Deutério/química , Liofilização , Humanos , Hidrogênio/química , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Sacarose/química , Temperatura de Transição , Trealose/químicaRESUMO
The goal of the work described in this publication was to evaluate a new, small, material-sparing freeze dryer, denoted as the "mini-freeze dryer or mini-FD", capable of reproducing the product temperature history of larger freeze dryers, thereby facilitating scale-up. The mini-FD wall temperatures can be controlled to mimic loading procedures and dryer process characteristics of larger dryers. The mini-FD is equipped with a tunable diode laser absorption spectroscopy (TDLAS) water vapor mass flow monitor and with other advanced process analytical technology (PAT) sensors. Drying experiments were performed to demonstrate scalability to larger freeze dryers, including the determination of vial heat transfer coefficients, K v . Product temperature histories during K v runs were evaluated and compared with those obtained with a commercial laboratory-scale freeze dryer (LyoStar II) for sucrose and mannitol product formulations. When the mini-FD wall temperature was set at the LyoStar II band temperature (- 20°C) to mimic lab dryer edge vials, edge vial drying in the mini-FD possessed an average K v within 5% of those obtained during drying in the LyoStar II. When the wall temperature of the mini-FD was set equal to the central vial product temperature, edge vials behaved as center vials, possessing a K v value within 5% of those measured in the LyoStar II. During both K v runs and complete product freeze drying runs, the temperature-time profiles for the average edge vials and central vial in the mini-FD agreed well with the average edge and average central vials of the LyoStar II.
Assuntos
Liofilização/instrumentação , Liofilização/métodos , Temperatura Alta , Manitol , Sacarose , TemperaturaRESUMO
Inter- and intra-batch variability in heat and mass transfer during the drying phase of lyophilization is well recognized. Heat transfer variability between individual vials in the same batch arise from both different positions in the vial array and from variations in the bottom contour of the vials, both effects contributing roughly equally to variations in the effective heat transfer coefficient of the vials, Kv. Both effects can be measured in the laboratory, and variations in average Kv values as a function of vial position in the array for lab and production can be calculated by use of the simple steady-state heat and mass transfer theory. Typically, in the laboratory dryer, vials on the edge of the array, "edge vials," run 2-4°C warmer than "center vials," but differences between laboratory and manufacturing temperatures are modest. The variability in mass transfer can be assigned to major variations in ice nucleation temperature (both intra-batch and inter-batch), including major differences between laboratory and manufacturing. The net effect of all random variations, for each class of vial, can be evaluated by a simple statistical model-propagation of error, which then allows prediction of the distribution in product temperatures and drying times, and therefore prediction of percent of vials dry and percent of vials collapsed and proximity to the edge of failure for a given process. Good agreement between theoretical and experimentally determined maximum temperatures in primary drying and percent collapsed product demonstrates the calculations have useful accuracy.
Assuntos
Embalagem de Medicamentos/métodos , Liofilização/métodos , Temperatura Alta , Tecnologia Farmacêutica/métodos , Embalagem de Medicamentos/normas , Liofilização/normas , Vidro/normas , Peso Molecular , Tecnologia Farmacêutica/normasRESUMO
Product temperature during the primary drying step of freeze-drying is controlled by a set point chamber pressure and shelf temperature. However, recent computational modeling suggests a possible variation in local chamber pressure. The current work presents an experimental verification of the local chamber pressure gradients in a lab-scale freeze-dryer. Pressure differences between the center and the edges of a lab-scale freeze-dryer shelf were measured as a function of sublimation flux and clearance between the sublimation front and the shelf above. A modest 3-mTorr difference in pressure was observed as the sublimation flux was doubled from 0.5 to 1.0 kg·h-1·m-2 at a clearance of 2.6 cm. Further, at a constant sublimation flux of 1.0 kg·h-1·m-2, an 8-fold increase in the pressure drop was observed across the shelf as the clearance was decreased from 4 to 1.6 cm. Scale-up of the pressure variation from lab- to a manufacturing-scale freeze-dryer predicted an increased uniformity in drying rates across the batch for two frequently used pharmaceutical excipients (mannitol and sucrose at 5% w/w). However, at an atypical condition of shelf temperature of +10°C and chamber pressure of 50 mTorr, the product temperature in the center vials was calculated to be a degree higher than the edge vial for a low resistance product, thus reversing the typical edge and center vial behavior. Thus, the effect of local pressure variation is more significant at the manufacturing-scale than at a lab-scale and accounting for the contribution of variations in the local chamber pressures can improve success in scale-up.
Assuntos
Tecnologia Farmacêutica/métodos , Excipientes/química , Liofilização/métodos , Manitol/química , Pressão , Sacarose/química , Temperatura , Água/químicaRESUMO
The flow physics in the product chamber of a freeze dryer involves coupled heat and mass transfer at different length and time scales. The low-pressure environment and the relatively small flow velocities make it difficult to quantify the flow structure experimentally. The current work presents the three-dimensional computational fluid dynamics (CFD) modeling for vapor flow in a laboratory scale freeze dryer validated with experimental data and theory. The model accounts for the presence of a non-condensable gas such as nitrogen or air using a continuum multi-species model. The flow structure at different sublimation rates, chamber pressures, and shelf-gaps are systematically investigated. Emphasis has been placed on accurately predicting the pressure variation across the subliming front. At a chamber set pressure of 115 mtorr and a sublimation rate of 1.3 kg/h/m2, the pressure variation reaches about 9 mtorr. The pressure variation increased linearly with sublimation rate in the range of 0.5 to 1.3 kg/h/m2. The dependence of pressure variation on the shelf-gap was also studied both computationally and experimentally. The CFD modeling results are found to agree within 10% with the experimental measurements. The computational model was also compared to analytical solution valid for small shelf-gaps. Thus, the current work presents validation study motivating broader use of CFD in optimizing freeze-drying process and equipment design.
Assuntos
Desenho de Equipamento/métodos , Liofilização/métodos , Simulação por Computador , Temperatura Alta , Modelos Teóricos , PressãoRESUMO
Recommended best practices in monitoring of product status during pharmaceutical freeze drying are presented, focusing on methods that apply to both laboratory and production scale. With respect to product temperature measurement, sources of uncertainty associated with any type of measurement probe are discussed, as well as important differences between the two most common types of temperature-measuring instruments-thermocouples and resistance temperature detectors (RTD). Two types of pressure transducers are discussed-thermal conductivity-type gauges and capacitance manometers, with the Pirani gauge being the thermal conductivity-type gauge of choice. It is recommended that both types of pressure gauge be used on both the product chamber and the condenser for freeze dryers with an external condenser, and the reasoning for this recommendation is discussed. Developing technology for process monitoring worthy of further investigation is also briefly reviewed, including wireless product temperature monitoring, tunable diode laser absorption spectroscopy at manufacturing scale, heat flux measurement, and mass spectrometry as process monitoring tools.
Assuntos
Liofilização/instrumentação , Tecnologia Farmacêutica/instrumentação , Liofilização/métodos , Pressão , Análise Espectral , Tecnologia Farmacêutica/métodos , TemperaturaRESUMO
Raman scattering shows promise as a powerful routine tool, to determine both secondary and the smaller tertiary structural changes that precede aggregation in both solutions and solids. A method was developed utilizing principal component analysis (PCA) of Raman spectra for detection of small, but meaningful, pH induced changes in tertiary protein structure linked to aggregate formation using α-lactalbumin solutions as a model. The sample preparation and spectral parameters, were optimized for a bulk Raman probe. Analysis of large regions (600-1850 cm-1) yielded principal component (PC) scores useful for semi-quantitative comparison of protein conformation between formulations. PC loadings corresponded to specific structural peaks known to change with solution pH. PCA of circular dichroism (CD) spectra of dilute solutions yielded similar results. Sucrose is a common formulation excipient with a Raman spectrum that overlaps many protein peaks. With sucrose in the protein solution, the ability of PCA to discern protein structural changes from the Raman spectra was somewhat reduced. Analysis of a more limited spectral region (1530-1780 cm-1) with negligible sucrose spectral contribution improved the discrimination of protein conformational states. The new Raman method accurately distinguished differences in protein structure in concentrated solutions. The long-term goal is to explore Raman characterization as a routine monitoring tool of protein stability in both solution and solid states.
Assuntos
Quimiometria , Proteínas , Conformação Proteica , Excipientes/química , Estrutura Terciária de Proteína , Análise Espectral Raman/métodosRESUMO
PURPOSE: To relate NMR relaxation times to instability-related molecular motions of freeze-dried protein formulations and to examine the effect of sugars on these motions. METHODS: Rotating-frame spin-lattice relaxation time (T(1ρ)) was determined for both protein and sugar carbons in freeze-dried lysozyme-sugar (trehalose, sucrose and isomaltose) formulations using solid-state (13)C NMR. RESULTS: The temperature dependence of T(1ρ) for the lysozyme carbonyl carbons in lysozyme with and without sugars was describable with a model that includes two different types of molecular motion with different correlation times (τ(c)) for the carbon with each τ(c) showing Arrhenius temperature dependence. Both relaxation modes have much smaller relaxation time constant (τ(c)) and temperature coefficient (Ea) than structural relaxation and may be classified as ß-relaxation and γ-relaxation. The τ(c) and Ea for γ-relaxation were not affected by sugars, but those for ß-relaxation were increased by sucrose, changed little by trehalose, and decreased by isomaltose, suggesting that the ß-mobility of the lysozyme carbonyl carbons is decreased by sucrose and increased by isomaltose. CONCLUSION: T(1ρ) determined for the lysozyme carbonyl carbons can reflect the effect of sugars on molecular mobility in lysozyme. However, interpretation of relaxation time data is complex and may demand data over an extended temperature range.
Assuntos
Isomaltose/química , Muramidase/química , Sacarose/química , Trealose/química , Liofilização , Espectroscopia de Ressonância Magnética , Movimento (Física) , TemperaturaRESUMO
Measurement of drug solubility is one of the key elements of active pharmaceutical ingredient (API) characterization during the drug discovery and development process. This report is a critical review of experimental methods reported in the literature for the measurement of aqueous solubility of amorphous, partially crystalline and crystalline organic compounds. A summary of high-throughput automated methods used in early drug discovery research is also provided in this report. This review summarizes the challenges that are encountered during solubility measurement and the complexities that are often overlooked. Even though there is an advantage in using the amorphous form of a drug due to its higher solubility, measurement of its solubility with useful accuracy is still a practical problem. Therefore, this review provides recommendations of preferred methods and precautions in using these methods to determine the aqueous solubility of amorphous and crystalline new molecular entities, with emphasis on the physico-chemical characterization of the solid state of the test substance.
Assuntos
Química Farmacêutica/métodos , Descoberta de Drogas/métodos , Preparações Farmacêuticas/química , Fenômenos Químicos , Cristalização , Estabilidade de Medicamentos , Transição de Fase , Solubilidade , Termodinâmica , Água/químicaRESUMO
A case study has been developed to illustrate one way of incorporating a Quality by Design approach into formulation and process development for a small molecule, freeze-dried parenteral product. Sodium ethacrynate was chosen as the model compound. Principal degradation products of sodium ethacrynate result from hydrolysis of the unsaturated ketone in aqueous solution, and dimer formation from a Diels-Alder condensation in the freeze-dried solid state. When the drug crystallizes in a frozen solution, the eutectic melting temperature is above -5°C. Crystallization in the frozen system is affected by pH in the range of pH 6-8 and buffer concentration in the range of 5-50 mM, where higher pH and lower buffer concentration favor crystallization. Physical state of the drug is critical to solid state stability, given the relative instability of amorphous drug. Stability was shown to vary considerably over the ranges of pH and buffer concentration examined, and vial-to-vial variability in degree of crystallinity is a potential concern. The formulation design space was constructed in terms of pH and drug concentration, and assuming a constant 5 mM concentration of buffer. The process design space is constructed to take into account limitations on the process imposed by the product and by equipment capability.
Assuntos
Desenho de Fármacos , Ácido Etacrínico/química , Soluções Tampão , Química Farmacêutica , Cristalização , Estabilidade de Medicamentos , Liofilização , Concentração de Íons de Hidrogênio , Hidrólise , Temperatura de TransiçãoRESUMO
Although several guidelines do exist for freeze-drying process development and scale-up, there are still a number of issues that require additional attention. The objective of this review article is to discuss some emerging process development and scale-up issue with emphasis on effect of load condition and freeze-drying in novel container systems such as syringes, Lyoguard trays, ampoules, and 96-well plates. Understanding the heat and mass transfer under different load conditions and for freeze-drying in these novel container systems will help in developing a robust freeze-drying process which is also easier to scale-up. Further research and development needs in these emerging areas have also been addressed.
Assuntos
Embalagem de Medicamentos/métodos , Liofilização/métodos , Esterilização/métodos , Temperatura Alta , SeringasRESUMO
The secondary drying phase in freeze drying is mostly developed on a trial-and-error basis due to the lack of appropriate noninvasive process analyzers. This study describes for the first time the application of Tunable Diode Laser Absorption Spectroscopy, a spectroscopic and noninvasive sensor for monitoring secondary drying in laboratory-scale freeze drying with the overall purpose of targeting intermediate moisture contents in the product. Bovine serum albumin/sucrose mixtures were used as a model system to imitate high concentrated antibody formulations. First, the rate of water desorption during secondary drying at constant product temperatures (-22 °C, -10 °C, and 0 °C) was investigated for three different shelf temperatures. Residual moisture contents of sampled vials were determined by Karl Fischer titration. An equilibration step was implemented to ensure homogeneous distribution of moisture (within 1%) in all vials. The residual moisture revealed a linear relationship to the water desorption rate for different temperatures, allowing the evaluation of an anchor point from noninvasive flow rate measurements without removal of samples from the freeze dryer. The accuracy of mass flow integration from this anchor point was found to be about 0.5%. In a second step, the concept was successfully tested in a confirmation experiment. Here, good agreement was found for the initial moisture content (anchor point) and the subsequent monitoring and targeting of intermediate moisture contents. The present approach for monitoring secondary drying indicated great potential to find wider application in sterile operations on production scale in pharmaceutical freeze drying.
Assuntos
Liofilização , Preparações Farmacêuticas , Análise Espectral , Absorção , Animais , Bovinos , Dessecação , Liofilização/instrumentação , Liofilização/métodos , Soroalbumina Bovina , Análise Espectral/instrumentação , Análise Espectral/métodos , Sacarose/química , Tecnologia Farmacêutica , Temperatura , Água/análiseRESUMO
PURPOSE: To quantitatively assess the solubility advantage of amorphous forms of nine insoluble drugs with a wide range of physico-chemical properties utilizing a previously reported thermodynamic approach. METHODS: Thermal properties of amorphous and crystalline forms of drugs were measured using modulated differential calorimetry. Equilibrium moisture sorption uptake by amorphous drugs was measured by a gravimetric moisture sorption analyzer, and ionization constants were determined from the pH-solubility profiles. Solubilities of crystalline and amorphous forms of drugs were measured in de-ionized water at 25°C. Polarized microscopy was used to provide qualitative information about the crystallization of amorphous drug in solution during solubility measurement. RESULT: For three out the nine compounds, the estimated solubility based on thermodynamic considerations was within two-fold of the experimental measurement. For one compound, estimated solubility enhancement was lower than experimental value, likely due to extensive ionization in solution and hence its sensitivity to error in pKa measurement. For the remaining five compounds, estimated solubility was about 4- to 53-fold higher than experimental results. In all cases where the theoretical solubility estimates were significantly higher, it was observed that the amorphous drug crystallized rapidly during the experimental determination of solubility, thus preventing an accurate experimental assessment of solubility advantage. CONCLUSION: It has been demonstrated that the theoretical approach does provide an accurate estimate of the maximum solubility enhancement by an amorphous drug relative to its crystalline form for structurally diverse insoluble drugs when recrystallization during dissolution is minimal.
Assuntos
Preparações Farmacêuticas/química , Termodinâmica , Cromatografia Líquida de Alta Pressão , Modelos Químicos , Estrutura Molecular , SolubilidadeRESUMO
Freeze-drying is a relatively expensive process requiring long processing time, and hence one of the key objectives during freeze-drying process development is to minimize the primary drying time, which is the longest of the three steps in freeze-drying. However, increasing the shelf temperature into secondary drying before all of the ice is removed from the product will likely cause collapse or eutectic melt. Thus, from product quality as well as process economics standpoint, it is very critical to detect the end of primary drying. Experiments were conducted with 5% mannitol and 5% sucrose as model systems. The apparent end point of primary drying was determined by comparative pressure measurement (i.e., Pirani vs. MKS Baratron), dew point, Lyotrack (gas plasma spectroscopy), water concentration from tunable diode laser absorption spectroscopy, condenser pressure, pressure rise test (manometric temperature measurement or variations of this method), and product thermocouples. Vials were pulled out from the drying chamber using a sample thief during late primary and early secondary drying to determine percent residual moisture either gravimetrically or by Karl Fischer, and the cake structure was determined visually for melt-back, collapse, and retention of cake structure at the apparent end point of primary drying (i.e., onset, midpoint, and offset). By far, the Pirani is the best choice of the methods tested for evaluation of the end point of primary drying. Also, it is a batch technique, which is cheap, steam sterilizable, and easy to install without requiring any modification to the existing dryer.
Assuntos
Liofilização/métodos , Dessecação , Umidade , Gelo/análise , Manitol/análise , Manometria , Pressão , Análise Espectral , Sacarose/análise , Sacarose/química , Temperatura , Água/análiseRESUMO
Controlling ice nucleation, at a fixed higher temperature, results in larger ice crystals, which can reduce the ice/freeze-concentrate interface area where proteins can adsorb and partially unfold. Moreover, limited work has been done to address any effects on short-term stability due to a slow ramp or long isothermal hold after the ice nucleation step. The objective was to evaluate the effect of the ice nucleation temperature and residence time in the freeze-concentrate on in-process or storage stability of representative proteins, human IgG, and recombinant human serum albumin. The results suggest a higher ice nucleation temperature can minimize aggregation of protein pharmaceuticals, which are labile at ice/aqueous interface. Apart from the ice nucleation step, the present study identified the residence time in the freeze-concentrate as the critical factor that influences protein stability post ice nucleation. At a temperature where enough mobility exists (i.e., above Tg' of the formulation), the long residence time in the freeze-concentrate can result in significant protein aggregation during the process. In addition to stability, the findings revealed that not only the ice nucleation temperature but also the thermal history of the formulation post ice nucleation defines the surface area of ice and the porous structure of the freeze-dried cake.
Assuntos
Gelo , Liofilização , Congelamento , Humanos , Estabilidade Proteica , TemperaturaRESUMO
Quality by Design (QbD), aims at assuring quality by proper design and control, utilizing appropriate Process Analytical Technologies (PAT) to monitor critical process parameters during processing to ensure that the product meets the desired quality attributes. This review provides a comprehensive list of process monitoring devices that can be used to monitor critical process parameters and will focus on a critical review of the viability of the PAT schemes proposed. R&D needs in PAT for freeze-drying have also been addressed with particular emphasis on batch techniques that can be used on all the dryers independent of the dryer scale.
Assuntos
Técnicas de Química Analítica/métodos , Liofilização/métodos , Tecnologia Farmacêutica/métodos , Técnicas de Química Analítica/instrumentação , Liofilização/instrumentação , Controle de Qualidade , Tecnologia Farmacêutica/instrumentaçãoRESUMO
The purpose of this paper was to identify an optimal formulation, free of any human or animal derived protein, which stabilizes biosynthetic Factor VIII (rAHF) during freeze drying and storage. Factor VIII activity in samples stored at temperatures between 25 degrees C and 60 degrees C was determined using the one-stage activated partial thromboplastin time assay. Various formulations containing different combinations of a stabilizer and a bulking agent were screened for acceptable freeze-drying behavior, elegance of the resulting product, and stability during processing and storage. Degradation of freeze-dried rAHF followed the 'square root of time' kinetics. Stability of rAHF was found to increase with increasing protein concentration, indicating a self-protection effect. The addition of the antioxidant, glutathione was also shown to enhance storage stability. Given the constraint of high residual levels of NaCl from purification, the lead formulation employed mannitol as a bulking agent and trehalose as the general stabilizer. This formulation allowed an elegant product to be produced which more than met the stability requirements. However, it was also shown that elimination of residual NaCl allowed a much shorter freeze-drying cycle to produce an elegant product with greatly enhanced stability.
Assuntos
Química Farmacêutica/métodos , Fator VIII/biossíntese , Fator VIII/química , Alanina/química , Antioxidantes/química , Arginina/química , Varredura Diferencial de Calorimetria , Estabilidade de Medicamentos , Fator VIII/metabolismo , Liofilização/métodos , Glutationa/química , Glicerol/química , Glicina/química , Histidina/química , Derivados de Hidroxietil Amido/química , Cinética , Manitol/química , Oxirredução , Tempo de Tromboplastina Parcial , Estabilidade Proteica , Rafinose/química , Cloreto de Sódio/química , Sacarose/química , Temperatura , Temperatura de Transição , Trealose/química , Difração de Raios XRESUMO
A method to achieve controlled ice nucleation during the freeze-drying process using an ice fog technique was demonstrated in an earlier report. However, the time required for nucleation was about 5 min, even though only one shelf was used, which resulted in Ostwald ripening (annealing) in some of the vials that nucleated earlier than the others. As a result, the ice structure was not optimally uniform in all the vials. The objective of the present study is to introduce a simple variation of the ice fog method whereby a reduced pressure in the chamber is utilized to allow more rapid and uniform freezing which is also potentially easier to scale up. Experiments were conducted on a lab scale freeze dryer with sucrose as model compound at different concentration, product load, and fill volume. Product resistance during primary drying was measured using manometric temperature measurement. Specific surface area of the freeze-dried cake was also determined. No difference was observed either in average product resistance or specific surface area for the different experimental conditions studied, indicating that with use of the reduced pressure ice fog technique, the solutions nucleated at very nearly the same temperature (-10 degrees C). The striking feature of the "Reduced Pressure Ice Fog Technique" is the rapid ice nucleation (less than a minute) under conditions where the earlier procedure required about 5 min; hence, effects of variable Ostwald ripening were not an issue.