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Influence of controlled nucleation by ultrasounds on ice morphology of frozen formulations for pharmaceutical proteins freeze-drying
Chemical Engineering and Processing 45 (2006) 783–791
Influence of controlled nucleation by ultrasounds on ice morphology of frozen formulations for pharmaceutical proteins freeze-drying Kyuya Nakagawa, Aur´elie Hottot, S´everine Vessot, Julien Andrieu ∗ Laboratoire d’Automatique et de G´enie des Proc´ed´es (LAGEP), UMR Q 5007 CNRS UCB Lyon1-CPE, Bˆat. 308G, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France Received 22 November 2005; received in revised form 10 March 2006; accepted 10 March 2006 Available online 17 March 2006
Abstract In order to optimize industrial freeze-drying processes of pharmaceutical proteins in vials, an ultrasound system has been designed and applied to control the freezing step. It was confirmed that during supercooling, nucleation temperatures of the sample could be controlled at selected values below the equilibrium freezing temperature. Because the main freezing elementary phenomena like nucleation and ice crystals growth are strongly related with ice crystal morphology parameters, the controlled nucleation by ultrasound effectively modified notably the primary drying duration. Thus, it was experimentally confirmed that the primary drying rates during freeze-drying of pharmaceutical formulations (i.e. mannitol, BSA, sucrose) were accelerated due to ice phase morphological modifications induced by the ultrasound control system. © 2006 Elsevier B.V. All rights reserved. Keywords: Controlled nucleation; Ultrasound; Freeze-drying; Ice crystal morphology
1. Introduction In order to reduce the operating costs related to pretty long drying times encountered during industrial freeze-drying process, morphological parameters of frozen phase need to be controlled and optimized. For this purpose, the freezing step control is an important challenge because the freezing elementary phenomena like nucleation and ice crystals growth are strongly related with ice crystal morphology parameters, thus resulting in consequent large distributions of primary drying rates. However, ice nucleation process is well known to be a spontaneous and stochastic phenomenon more or less related to material and process parameters that are generally difficult to control like impurities, asperities, surface roughness, etc. Nevertheless, Searles et al. in the study of freeze-drying of aqueous standard diluted formulations in vials used as model systems for pharmaceuticals proteins freeze-drying experimentally observed that some ice structure characteristic parameters and, by the way, primary drying rates were quite correlated to nucleation temper-
∗
Corresponding author. Tel.: +33 4 72 43 18 43; fax: +33 4 72 43 16 82. E-mail addresses: [email protected] (K. Nakagawa), [email protected] (J. Andrieu). 0255-2701/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cep.2006.03.007
atures i.e. to undercooling degrees and to thermal gradients in the system [1]. As a matter of fact, the undercooling degree of the solution determines the number of nuclei and, consequently, determines the ice crystals morphology in the sample [2]. Thus, it was previously observed that this structure and shape of ice crystals has a strong influence on the primary drying rates [3–5]. Thus, the control of the nucleation step appears to be a key factor for the optimization of industrial freeze-drying cycles of pharmaceuticals in vials. Furthermore, it has been before observed that ultrasounds were quite efficient to activate and to control the nucleation step during crystallization of aqueous solutions in agitated systems. Although the detailed mechanisms of nucleation of solid crystal from liquid solution under the influence of ultrasounds (i.e. sonocrystallization) are not still well elucidated, Ohsaka et al. experimentally proved that bubble cavitation produced by ultrasounds plays the main role on nuclei formation processes [6]. Besides, Hozumi et al. suggested that not only a single cavitation bubble but also a dipped metal bar worked as an effective nucleation site under the influence of ultrasound wave [7]. Inada et al. also reported the adequacy of sonication systems to control the phase change from supercooled water to ice formation [8] and Chow et al. precisely investigated the effects of ultrasounds during crystallization of sucrose solutions [9]. Consequently, all
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Fig. 1. The cooling device coupled with sonication system.
these data suggested the applicability of ultrasounds method for triggering the ice nucleation step and for controlling the ice crystal morphology during optimization of pharmaceuticals freeze-drying cycles [10]. In the present paper, an ultrasound system that allows the control of the nucleation step during the freeze-drying process of model formulations in commercial glass vials is described and the effect of the ultrasound on ice crystal nucleation is discussed. The ice crystal morphology was directly characterized by the optical microscopy method in the cold chamber, and the induced morphological modifications are presented and discussed. The influence of the nucleation temperature on the overall primary drying rates during standard cycles encountered in pharmaceutical protein freeze-drying is also presented. 2. Materials and method 2.1. Materials Three types of standard simplified formulations generally selected for heat sensitive pharmaceutical proteins freeze-drying were successively investigated, namely: • 10% mannitol solution prepared from distilled water and mannitol powder (Fluka Chemie AG) which corresponded to a crystalline system;
• 5% BSA (bovin serum albumin) type formulation (Sigma– Aldrich) which corresponded to an amorphous system; • 10% sucrose solution, extensively used in industrial protein freeze-drying, and prepared from distilled water and sucrose powder (PROLABO) which also corresponded to an amorphous system. 2.2. System of freezing under ultrasound controlled nucleation in vials The cooling stage combined with ultrasound transducer was specially manufactured by SODEVA Company (France) in order to trigger and to control the nucleation process of the system as schematize on Fig. 1. An ultrasound transducer was tightly attached to an aluminium plate (200 mm × 200 mm, 5 mm thickness) and, as shown on Fig. 1A, by clamping the aluminium plate itself to a horn of the transducer. Furthermore, the same transducer was coupled with an ultrasound generator (MW400GSIP, SODEVA, France) and the frequency of the vibration was tuned up around 35.89 kHz in order to obtain an adequate frequency of resonance on the aluminium plate. The aluminium plate attached to the transducer was placed in thermal contact as close as possible with the aluminium heat exchanger (width 300 mm, length 100 mm, height 10 mm) by clamper fixing. A cooling fluid (Therminol D12, Solutia) was circulated through the heat exchanger at precisely controlled
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temperatures with a thermo regulated cryofluid bath (CC180, Huber, Germany). Then, the liquid solution samples were introduced inside the vials and the vials were placed on the aluminium plate. Due to ultrasound vibrations on the plate and to resonance phenomena, nodal points existed all through the plate surface. As sketched in Fig. 1C, the nodal points were observed by setting a powder layer on the aluminium plates, and then propagating ultrasound. The powder on the nodal points does not move on the surface, and there is no splashing off the surface. Thus, the places of these nodal points should be avoided for the vial setting because, at these points, the ultrasound vibrations could not propagate at all inside the vial. Besides, to enhance this propagation, a thin layer of silicon oil was inserted between the curved bottom of a vial and the aluminium plate (Fig. 1B), because without this contact fluid the ultrasound waves could not efficiently propagate all through the whole vial solution and, furthermore, in absence of silicon oil, vibration causes the vials to slip and eventually be overturned; the presence of silicon oil hinders such undesired effects. The nucleation of the samples was realized under the following freezing protocol: • samples stabilization on the cold stage for 15 min at 5 ◦ C; • starting cooling at −1 ◦ C/min; • at selected temperature, triggering ultrasound wave during about 1 s at frequency of 35.89 kHz with an electric output of 40 W (controlled nucleation); • continuing cooling down to −40 ◦ C (−60 ◦ C for 10% sucrose solution) at −1 ◦ C/min up to achieve complete sample solidification. This electric output (40 W) at the frequency of 35.89 KHz was determined from trial and error runs to be able to transmit efficiently the ultrasound waves to the system. Tubing glass vials (Verretubex, France) of 3 mL (vial diameter D = 12 mm) were used in this work. In order to avoid undesirable impurities, the vials were washed as clean as possible, with an ultrasound bath during 10 min with distilled water, dried by compressed air and then, immediately stoppered. Sample solution (0.75 mL) was filled in each vial (solution height h = 7 mm) and the sample temperatures were monitored by type K thermocouples. Sensitive extremities of the thermocouples were attached by pads (COMARK) on the exterior surface of each vial wall, at middle position between liquid meniscus and vial bottom i.e. at around 4 mm height from the vial bottom. By this external non invasive fixing, the nucleation phenomena was not submitted to any artifact that usually results from introduction of the thermocouples inside the vial; besides, an adequate thermal paste (TECHSPRAY) was used for having a better thermal contact between thermocouple sensitive part and the external surface of the vial glass wall. Sample temperatures were stored and recorded with a data acquisition system (4100G Eurotherm Recorders) every 2 s. 2.3. Direct optical microscopy observation method The ice crystal morphology was analyzed by a photonic microscopy method by reflexion with episcopic coaxial lighting,
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Fig. 2. Principle of photonic microscopy with episcopic coaxial lighting.
the principle of which is schematized in Fig. 2. This method is essentially based on the light flux reflected by the surface of the sample and has the main advantage of preserving well the original structure of the frozen sample as proposed by Faydi et al. in the case of frozen foods (ice creams) structure analysis [11,12]. Ueno et al. developed this technique and successively obtained three-dimensional ice crystal images [13]. The direct observation was realized in a cold chamber maintained at −25 ◦ C where the microtome (LEICA 2000R), the microscope (LEICA MZ12), the digital camera (Hitachi CCD) and an optical fibre providing the episcopic coaxial lighting were placed. A frozen sample prepared as described above was taken out of the vial by carefully crashing the vial, then fixing it on a metal support by tissue freezing medium (LEICA Instruments GmbH) and finally stored in liquid nitrogen. Before the observation, the sample was cut and carefully smoothed down to obtain a surface roughness lower than 1 m. The obtained images were numerically stored in the computer and then were analyzed by Image J 1.34 software that allowed the estimation of the equivalent and mean particle sizes. 2.4. Freeze-drying procedures Frozen samples were freeze-dried (sublimation period) with a laboratory pilot freeze-dryer (USIFROID SMH45, France) using the following protocol [14,15]: • vials with frozen samples placed on the pre-cooled shelf at Ts = −40 ◦ C (−60 ◦ C for 10% sucrose solution) in the freezedryer chamber; • vacuum setting inside the freeze-dryer up to Pc = 10 Pa; • increase the shelf temperature from −40 to −20 ◦ C (from −60 to −40 ◦ C for sucrose solution) at +1 ◦ C/min; • maintain steady freeze-drying conditions all along the primary drying period. A set of 30 vials was put in the same place of the plate of the freeze-dryer for each freeze-drying experiment, and two or three runs were repeated for each experimental condition. In order to determine the drying curves, samples were taken out at the fixed durations with a thief door without breaking vacuum in the chamber, and the weight loss was measured, from which the drying curves were calculated. In order to determine the primary drying rates, the freeze-drying run was stopped after 3–4 h of sublimation, their duration corresponding to about 30–40 wt.% of total ice sublimed.
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3. Results and discussions 3.1. Nucleation temperatures It is well known that supercooled solutions with any control system start their nucleation quite randomly so that, for the same cooling rate and the same experimental conditions, the corresponding nucleation temperatures are largely distributed. From our experiment, the nucleation temperatures were experimentally determined from the temperature profiles recording data, at the minimum point of the undercooling curve when the temperature increases again (due to exothermic nuclei formation) up to the equilibrium freezing temperature. Thus, Fig. 3 shows the frequency distribution of the experimental spontaneous nucleation temperatures for the mannitol, BSA and sucrose systems frozen at the same cooling rate equal to −1 ◦ C/min. These data showed that the nucleation temperatures varied from −2 to −15 ◦ C for all the systems with averaged values equal to −8 ◦ C. All these data were obtained in similar rigorously controlled cooling conditions (cooling rates) and with vials (type, size, cleanness, etc.) as clean and identical as possible. The nucleation of the sample was triggered at selected temperature levels lower than equilibrium freezing temperature. Fig. 4 illustrates the freezing curves obtained with 10% mannitol solutions nucleated by the previously described ultrasound system. When the nucleation process was triggered at low temperature levels corresponding to high undercooling levels, the solution solidified immediately. However, if the nucleation processes was triggered at lower undercooling levels, for example above −3.5 ◦ C, the system was first transformed into a kind of slurry and then reached more gradually the frozen state. First, we confirmed experimentally that the investigated experimented sonication system was useful to trigger the nucleation process of the supercooled solution contained in the vial with a high degree of repeatability. However, it was sometimes observed that some
Fig. 3. Frequency distributions of spontaneous nucleation temperatures.
Fig. 4. Freezing curves of ultrasounds nucleated samples (10% mannitol).
vials did not respond at all to the ultrasound pulse. After more precise observation, it approved that these vials were not placed in stable position on the plates due to the roughness of their bottom or for other unknown reasons, so that the ultrasound wave could not efficiently propagate throughout the whole volume of the vial. Furthermore, in some cases, we also experimentally observed that some samples without triggered nucleation could be nucleated by holding them tightly on the plate. So, it should be mentioned that good mechanical and thermal contact between the vial bottoms, which are quite curved, and the plate surface of the freeze-dryer must be achieved for implementing properly the present system. 3.2. Characterization of ice crystals structure Ice crystal morphologies of 10% mannitol frozen solutions were directly observed by the optical microscopy method in the cold chamber (−25 ◦ C) presented above. The surfaces observed by microscopy were vertical and horizontal sample cuts that corresponded to a cross section of a cylindrical sample frozen in vials. Some typical images that represented the more characteristic trends of each sample are selected and shown in Fig. 5 (vertical cross sections) and in Fig. 6 (horizontal cross sections). In these images, the ice crystals appear in white color and the cryoconcentrated phase, principally corresponding to mannitol crystals, in black colour. One can see in these figures obvious morphological differences of ice crystals structure depending on their nucleation temperatures. Large and directional ice crystals (dendrite type) were confirmed in the sample nucleated at higher temperatures i.e. with lower supercooling degrees, while small and numerous heterogeneous ice crystals appeared in the sample nucleated at lower temperature i.e. with higher supercooling degree. Ice crystal growth in a vial generally starts from the bottom of the vial and progresses to the top, while a highly cryoconcentrated solution layer is formed at the top of the sam-
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Fig. 5. Ice crystal morphologies on vertical cross sections (10% mannitol): (A) upper position, nucleated at −2.04 ◦ C; (B) lower position, nucleated at −2.04 ◦ C; (C) upper position, nucleated at −8.17 ◦ C; (D) lower position, nucleated at −8.17 ◦ C.
ple. It is also confirmed on our vertical sample images (Fig. 5A and C) that the ice crystal initiation by the ultrasound waves occurred at the bottom of the vial, because smaller ice crystals were observed at the vial bottom than at the vial top. This fact was also confirmed by the comparison of ice crystal morphology images between a sample nucleated by ultrasound and a sample spontaneously nucleated at the same temperature. This means that the ice crystal growth nucleated by ultrasound progressed in
the same manner as in case of spontaneous nucleation. Thus, the average crystal size had almost the same value for the two types of nucleation as shown in Fig. 7. It is worth noting that there were no significant differences in the ice morphology of frozen sample prepared with or without ultrasound if they nucleated at the same temperature. Thus, one can say that the ice morphological parameters are mostly determined by the nucleation temperature values for the same operating conditions (cooling
Fig. 6. Ice crystal morphologies on horizontal cross sections (10% mannitol): (A) edge position, nucleated at −2.04 ◦ C; (B) center position, nucleated at −2.04 ◦ C; (C) edge position, nucleated at −7.39 ◦ C; (D) center position, nucleated at −7.39 ◦ C.
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Fig. 7. Ice crystal morphologies (10% mannitol): (1) spontaneous nucleation at −7.64 ◦ C; (2) controlled nucleation by ultrasound at −7.39 ◦ C. Table 1 Mean ice crystal equivalent diameters Nucleation temperature (◦ C)
Upper position (m)
Lower position (m)
Mean value (m)
−2.04 −7.39
142 57.3
103 50.5
122 54.1
rate, sample geometry, etc.). Large and dendrite type ice crystals were also observed in the horizontal cuts of the sample nucleated at higher temperature (Fig. 6A and B); however, they did not always correspond to directional structures like for images of vertical cuts. One can suppose that the three-dimensional image results from the aggregations of dendrite ice crystal in vertical direction. On the other hand, on the horizontal cuts of the sample nucleated at lower temperature, small and numerous ice crystals can be observed (Fig. 6C and D); furthermore, from the images of both vertical and horizontal cuts of the samples (Fig. 5C and D and 6C and D) we could not observe any regular directional ice crystal growth. Thus, it would be reasonable to imagine the three-dimensional structure of the sample nucleated at lower temperature as a network of irregular layers of approximately spherical ice crystals. Ice crystal size distributions calculated by image analysis are plotted on Fig. 8. Three different horizontal images were analyzed for determining each crystal size distribution. The average ice crystal size values are listed on Table 1. One can see that the average crystal size of the sample nucleated at −2.04 ◦ C is more than twice as large as the one resulting from nucleation at −7.39 ◦ C. It was observed from this figure that the ice crystal size distributions also depended on axial positions inside the vial;
larger ice crystals were observed at the upper positions, while smaller ice crystals were observed at the vial bottom. Furthermore, this tendency was confirmed very clearly with sample nucleated at higher temperature. This behaviour suggests that one could not always assume homogeneity of dried layer permeability in considering mass transfer during the drying steps of the freeze-drying process. Besides, from the freeze-drying tests using microbalance technique, Pikal et al. reported that the water vapour mass transfer resistance of the dried product layer was not directly proportional to its thickness [16]. 3.3. Primary drying rate during freeze-drying As shown in the previous sections, we could prepare frozen samples nucleated at selected temperatures, and we could confirm obvious dependency of the ice crystal morphology on their nucleation temperature. Those morphological modifications were expected to modify the mass transfer permeability of water vapour and contribute to accelerate primary drying rates during freeze-drying. Fig. 9 shows the ratio of water mass remained in the sample during primary drying step to the initial mass as a function of sublimation time. We compare in this figure the drying kinetics of a series of frozen samples nucleated, respectively, at around −2 and −8 ◦ C. The sublimation of the samples nucleated at lower temperature progressed at almost the same sublimation rate. Nevertheless, one can observe that the sublimation of the samples nucleated at higher temperature progressed in different manner. At the beginning of the sublimation, the sublimation rates were similar to the rates observed with samples nucleated at lower temperature; however, after two hours of sublimation, the sublimation rate slightly increased, but after five hours of sublimation, it gradually decreased. It is quite
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Fig. 8. Ice crystal size distributions (10% mannitol): (A) nucleated at −2.04 ◦ C; (B) nucleated at −7.39 ◦ C.
evident that these sublimation kinetics are directly related to the ice phase morphology, i.e. to the permeability of the dried layer. The overall primary drying rate values determined gravimetrically are plotted in Figs. 10–12 as a function of nucleation temperature. These plots show proportional relationship between the primary drying rate and the nucleation temperature for each data set, that is to say, the sublimation rate increases with the nucleation temperature. This is a coherent result in accordance with the image analysis data on the ice crystal morphology presented in the previous paragraph. As shown in Fig. 10, at a given temperature, the primary drying rate is approximately the same in presence and in absence of ultrasound. However, ultrasound allows for nucleation at much higher temperature to happen, which yields an increase in primary drying rate (60% e.g. between nucleation temperatures −8 and −2 ◦ C). In case of 10% sucrose freeze-drying (Fig. 12), the enhancement on sublimation rate was much larger: the sublimation rate as doubled by increasing the nucleation temperatures from −8 to −2 ◦ C. Nevertheless,
Fig. 9. Drying curves during freeze-drying (10% mannitol; Ts = −20 ◦ C; Pc = 10 Pa).
for sucrose we set primary drying shelf temperature lower than for the 10% mannitol formulation freeze-drying (i.e. −20 ◦ C for 10% mannitol instead of −40 ◦ C for 10% sucrose freeze-drying) in order to avoid collapse phenomenon. Due to the lower values of water vapour transfer potential during sublimation, the mass transfer was much more controlled by the structural parameters of the dried layers. In case of 5% BSA freeze-drying (Fig. 11), the average sublimation rates were higher than with other formulations, and, the enhancement of the sublimation rate was smaller. However, in this last case, we could also observe a clear relationship between the sublimation rate and the corresponding nucleation temperature. It is noteworthy that spontaneously nucleated samples were located on the same line as the controlled nucleation samples. This behaviour seems to indicate that the ice crystal growth mechanisms are the same for samples nucleated by ultrasound and the samples nucleated spontaneously. It is suggested that,
Fig. 10. Primary drying rates versus nucleation temperatures (10% mannitol). Open symbol: controlled nucleation by ultrasound; closed symbol: spontaneous nucleation.
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4. Conclusions
Fig. 11. Primary drying rates versus nucleation temperatures (5% BSA). Open symbol: controlled nucleation by ultrasound; closed symbol: spontaneous nucleation.
for a given system, the structural parameters of a frozen formulation are strongly dependent on the nucleation temperatures, so that this parameter is relevant for the permeability of dried layer during sublimation step. Thus, these data confirm the adequacy of an ultrasound system to control the nucleation processes and, by the way, to accelerate notably the primary sublimation rates during the freeze-drying process of pharmaceuticals in vials, improving the quality factors related to the structural properties (rehydration facility, water vapour permeability, etc.) and reducing the operating costs related to drying times.
An ultrasound system was set up and was investigated in order to optimize an industrial freeze-drying process in vials by controlling the freezing step. It was clearly observed that the ultrasound system could efficiently trigger the nucleation process of the three kinds of standard formulations investigated in a vial configuration largely used for pharmaceutical proteins freeze-drying in industry. The nucleation temperature of the samples could be controlled at selected values below the equilibrium freezing temperature, corresponding to different undercooling states for the ice crystals growth. Analysis of the ice structure morphology by direct optical microscopy in a cold chamber indicated that nucleation temperatures correspond to different undercooling states for ice crystals growth, that is to say: small and numerous ice crystals are obtained at lower nucleation temperature (higher supercooling degree), while large and directional ice crystals (dendrite type) are obtained at higher nucleation temperature (lower supercooling degree). These morphological modifications on ice crystals clearly impacted on water vapour permeability during the subsequent sublimation step. Consequently, it was proved that a strong correlation exists between the sublimation kinetics and the ice morphology. A significant correlation between the overall primary drying rates and the nucleation temperatures were confirmed (i.e. the drying rates increased with the nucleation temperatures), and, as a result, a great enhancement of their sublimation rates was achieved by increasing their nucleation temperatures from the average spontaneous nucleation temperature to around −2 ◦ C with ultrasound control. Furthermore, the ice crystal size distribution was also depending on axial position inside the vial as more larger ice crystals were observed in the upper part than at the vial bottom; thus, it was suggested that we might have to take into consideration heterogeneous dried layers for a better understanding of mass and heat transfer phenomena during sublimation step of freeze-drying process. Nevertheless, this intra-vial heterogeneity could be largely reduced by using annealing treatments as already found out in our laboratory [17]. Finally, it is expected that this ultrasound control system, after further studies, could be developed and applied for the optimization of pharmaceuticals freeze-drying cycles in industrial condition. Acknowledgements This work is supported by a grant from Japan Society for the Promotion of Science. References
Fig. 12. Primary drying rates versus nucleation temperatures (10% sucrose). Open symbol: controlled nucleation by ultrasound; closed symbol: spontaneous nucleation.
[1] J.A. Searles, J.F. Carpenter, T.W. Randolph, The ice nucleation temperature determines the primary drying rate of lyophilization for samples frozen on a temperature-controlled shelf, J. Pharm. Sci. 90 (2001) 860–871. [2] J. Swarbrick, J.C. Baylan (Eds.), Encyclopedia of Pharmaceutical Technology, Marcel Dekker, New York, 2001.
K. Nakagawa et al. / Chemical Engineering and Processing 45 (2006) 783–791 [3] L. Rey, J.C. May (Eds.), Freeze-drying/Lyophilization of Pharmaceutical and Biological Products, Marcel Dekker, New York, 1999. [4] M. Kochs, C.H. Korber, I. Heschel, B. Nunner, The influence of the freezing process on vapour transport during sublimation in vacuumfreeze-drying, Int. J. Heat Mass Transfer 34 (1991) 2395–2408. [5] M. Kochs, C.H. Korber, I. Heschel, B. Nunner, The influence of the freezing process on vapour transport during sublimation in vacuumfreeze-drying of macroscopic samples, Int. J. Heat Mass Transfer 36 (1993) 1727–1738. [6] K. Ohsaka, E.H. Trinh, Dynamic nucleation of ice induced by a single stable cavitation bubble, Appl. Phys. Lett. 73 (1998) 129–131. [7] T. Hozumi, A. Saito, S. Okawa, T. Matsui, Freezing phenomena of supercooled water under impacts of ultrasonic waves, Int. J. Refrig.Revue Int. du Froid 25 (2002) 948–953. [8] T. Inada, X. Zhang, A. Yabe, Y. Kozawa, Active control of phase change from supercooled water to ice by ultrasonic vibration 1. Control of freezing temperature, Int. J. Heat Mass Transfer 44 (2001) 4523–4531. [9] R. Chow, R. Blindt, R. Chivers, M. Povey, The sonocrystallisation of ice in sucrose solutions: primary and secondary nucleation, Ultrasonics 41 (2003) 595–604. [10] J.A. Searles, J.F. Carpenter, T.W. Randolph, Annealing to optimize the primary drying rate, reduce freezing-induced drying rate heterogeneity, and determine Tg’ in pharmaceutical lyophilization, J. Pharm. Sci. 90 (2001) 872–887.
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[11] E. Faydi, J. Andrieu, P. Laurent, Experimental study and modelling of the ice crystal morphology of model standard ice cream. Part I. Direct characterization method and experimental data, J. Food Eng. 48 (2001) 283–291. [12] A. Caillet, C. Cogne, J. Andrieu, P. Laurent, A. Rivoire, Characterization of ice cream structure by direct optical microscopy. Influence of freezing parameters, Food Sci. Technol.-Leb. 36 (2003) 743–749. [13] S. Ueno, G.S. Do, Y. Sagara, K. Kudoh, T. Higuchi, Three-dimensional measurement of ice crystals in frozen dilute solution, Int. J. Refrig. 27 (2004) 302–308. [14] A. Hottot, S. Vessot, J. Andrieu, A direct characterization method of the ice morphology. Relationship between mean crystals size and primary drying times of freeze-drying processes, Dry. Technol. 22 (2004) 2009–2021. [15] A. Hottot, S. Vessot, J. Andrieu, Determination of mass and heat transfer parameters during freeze-drying cycle of pharmaceutical products, PDA J. Pharm. Sci. Technol. 59 (2005) 138–153. [16] M.J. Pikal, S. Shah, D. Senior, J.E. Lang, Physical chemistry of freezedrying: measurement of sublimation rates for frozen aqueous solution by a microbalance technique, J. Pharm. Sci. 72 (1983) 635–650. [17] P. Chouvenc, S. Vessot, J. Andrieu, P. Vacus, Optimization of the freezedrying cycle: Adaptation of the pressure rise analysis model to noninstantaneous isolation valves, PDA J. Pharm. Sci. Technol. 59 (2005) 298–310.
Influence of controlled nucleation by ultrasounds on ice morphology of frozen formulations for pharmaceutical proteins freeze-drying Kyuya Nakagawa, Aur´elie Hottot, S´everine Vessot, Julien Andrieu ∗ Laboratoire d’Automatique et de G´enie des Proc´ed´es (LAGEP), UMR Q 5007 CNRS UCB Lyon1-CPE, Bˆat. 308G, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France Received 22 November 2005; received in revised form 10 March 2006; accepted 10 March 2006 Available online 17 March 2006
Abstract In order to optimize industrial freeze-drying processes of pharmaceutical proteins in vials, an ultrasound system has been designed and applied to control the freezing step. It was confirmed that during supercooling, nucleation temperatures of the sample could be controlled at selected values below the equilibrium freezing temperature. Because the main freezing elementary phenomena like nucleation and ice crystals growth are strongly related with ice crystal morphology parameters, the controlled nucleation by ultrasound effectively modified notably the primary drying duration. Thus, it was experimentally confirmed that the primary drying rates during freeze-drying of pharmaceutical formulations (i.e. mannitol, BSA, sucrose) were accelerated due to ice phase morphological modifications induced by the ultrasound control system. © 2006 Elsevier B.V. All rights reserved. Keywords: Controlled nucleation; Ultrasound; Freeze-drying; Ice crystal morphology
1. Introduction In order to reduce the operating costs related to pretty long drying times encountered during industrial freeze-drying process, morphological parameters of frozen phase need to be controlled and optimized. For this purpose, the freezing step control is an important challenge because the freezing elementary phenomena like nucleation and ice crystals growth are strongly related with ice crystal morphology parameters, thus resulting in consequent large distributions of primary drying rates. However, ice nucleation process is well known to be a spontaneous and stochastic phenomenon more or less related to material and process parameters that are generally difficult to control like impurities, asperities, surface roughness, etc. Nevertheless, Searles et al. in the study of freeze-drying of aqueous standard diluted formulations in vials used as model systems for pharmaceuticals proteins freeze-drying experimentally observed that some ice structure characteristic parameters and, by the way, primary drying rates were quite correlated to nucleation temper-
∗
Corresponding author. Tel.: +33 4 72 43 18 43; fax: +33 4 72 43 16 82. E-mail addresses: [email protected] (K. Nakagawa), [email protected] (J. Andrieu). 0255-2701/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cep.2006.03.007
atures i.e. to undercooling degrees and to thermal gradients in the system [1]. As a matter of fact, the undercooling degree of the solution determines the number of nuclei and, consequently, determines the ice crystals morphology in the sample [2]. Thus, it was previously observed that this structure and shape of ice crystals has a strong influence on the primary drying rates [3–5]. Thus, the control of the nucleation step appears to be a key factor for the optimization of industrial freeze-drying cycles of pharmaceuticals in vials. Furthermore, it has been before observed that ultrasounds were quite efficient to activate and to control the nucleation step during crystallization of aqueous solutions in agitated systems. Although the detailed mechanisms of nucleation of solid crystal from liquid solution under the influence of ultrasounds (i.e. sonocrystallization) are not still well elucidated, Ohsaka et al. experimentally proved that bubble cavitation produced by ultrasounds plays the main role on nuclei formation processes [6]. Besides, Hozumi et al. suggested that not only a single cavitation bubble but also a dipped metal bar worked as an effective nucleation site under the influence of ultrasound wave [7]. Inada et al. also reported the adequacy of sonication systems to control the phase change from supercooled water to ice formation [8] and Chow et al. precisely investigated the effects of ultrasounds during crystallization of sucrose solutions [9]. Consequently, all
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Fig. 1. The cooling device coupled with sonication system.
these data suggested the applicability of ultrasounds method for triggering the ice nucleation step and for controlling the ice crystal morphology during optimization of pharmaceuticals freeze-drying cycles [10]. In the present paper, an ultrasound system that allows the control of the nucleation step during the freeze-drying process of model formulations in commercial glass vials is described and the effect of the ultrasound on ice crystal nucleation is discussed. The ice crystal morphology was directly characterized by the optical microscopy method in the cold chamber, and the induced morphological modifications are presented and discussed. The influence of the nucleation temperature on the overall primary drying rates during standard cycles encountered in pharmaceutical protein freeze-drying is also presented. 2. Materials and method 2.1. Materials Three types of standard simplified formulations generally selected for heat sensitive pharmaceutical proteins freeze-drying were successively investigated, namely: • 10% mannitol solution prepared from distilled water and mannitol powder (Fluka Chemie AG) which corresponded to a crystalline system;
• 5% BSA (bovin serum albumin) type formulation (Sigma– Aldrich) which corresponded to an amorphous system; • 10% sucrose solution, extensively used in industrial protein freeze-drying, and prepared from distilled water and sucrose powder (PROLABO) which also corresponded to an amorphous system. 2.2. System of freezing under ultrasound controlled nucleation in vials The cooling stage combined with ultrasound transducer was specially manufactured by SODEVA Company (France) in order to trigger and to control the nucleation process of the system as schematize on Fig. 1. An ultrasound transducer was tightly attached to an aluminium plate (200 mm × 200 mm, 5 mm thickness) and, as shown on Fig. 1A, by clamping the aluminium plate itself to a horn of the transducer. Furthermore, the same transducer was coupled with an ultrasound generator (MW400GSIP, SODEVA, France) and the frequency of the vibration was tuned up around 35.89 kHz in order to obtain an adequate frequency of resonance on the aluminium plate. The aluminium plate attached to the transducer was placed in thermal contact as close as possible with the aluminium heat exchanger (width 300 mm, length 100 mm, height 10 mm) by clamper fixing. A cooling fluid (Therminol D12, Solutia) was circulated through the heat exchanger at precisely controlled
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temperatures with a thermo regulated cryofluid bath (CC180, Huber, Germany). Then, the liquid solution samples were introduced inside the vials and the vials were placed on the aluminium plate. Due to ultrasound vibrations on the plate and to resonance phenomena, nodal points existed all through the plate surface. As sketched in Fig. 1C, the nodal points were observed by setting a powder layer on the aluminium plates, and then propagating ultrasound. The powder on the nodal points does not move on the surface, and there is no splashing off the surface. Thus, the places of these nodal points should be avoided for the vial setting because, at these points, the ultrasound vibrations could not propagate at all inside the vial. Besides, to enhance this propagation, a thin layer of silicon oil was inserted between the curved bottom of a vial and the aluminium plate (Fig. 1B), because without this contact fluid the ultrasound waves could not efficiently propagate all through the whole vial solution and, furthermore, in absence of silicon oil, vibration causes the vials to slip and eventually be overturned; the presence of silicon oil hinders such undesired effects. The nucleation of the samples was realized under the following freezing protocol: • samples stabilization on the cold stage for 15 min at 5 ◦ C; • starting cooling at −1 ◦ C/min; • at selected temperature, triggering ultrasound wave during about 1 s at frequency of 35.89 kHz with an electric output of 40 W (controlled nucleation); • continuing cooling down to −40 ◦ C (−60 ◦ C for 10% sucrose solution) at −1 ◦ C/min up to achieve complete sample solidification. This electric output (40 W) at the frequency of 35.89 KHz was determined from trial and error runs to be able to transmit efficiently the ultrasound waves to the system. Tubing glass vials (Verretubex, France) of 3 mL (vial diameter D = 12 mm) were used in this work. In order to avoid undesirable impurities, the vials were washed as clean as possible, with an ultrasound bath during 10 min with distilled water, dried by compressed air and then, immediately stoppered. Sample solution (0.75 mL) was filled in each vial (solution height h = 7 mm) and the sample temperatures were monitored by type K thermocouples. Sensitive extremities of the thermocouples were attached by pads (COMARK) on the exterior surface of each vial wall, at middle position between liquid meniscus and vial bottom i.e. at around 4 mm height from the vial bottom. By this external non invasive fixing, the nucleation phenomena was not submitted to any artifact that usually results from introduction of the thermocouples inside the vial; besides, an adequate thermal paste (TECHSPRAY) was used for having a better thermal contact between thermocouple sensitive part and the external surface of the vial glass wall. Sample temperatures were stored and recorded with a data acquisition system (4100G Eurotherm Recorders) every 2 s. 2.3. Direct optical microscopy observation method The ice crystal morphology was analyzed by a photonic microscopy method by reflexion with episcopic coaxial lighting,
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Fig. 2. Principle of photonic microscopy with episcopic coaxial lighting.
the principle of which is schematized in Fig. 2. This method is essentially based on the light flux reflected by the surface of the sample and has the main advantage of preserving well the original structure of the frozen sample as proposed by Faydi et al. in the case of frozen foods (ice creams) structure analysis [11,12]. Ueno et al. developed this technique and successively obtained three-dimensional ice crystal images [13]. The direct observation was realized in a cold chamber maintained at −25 ◦ C where the microtome (LEICA 2000R), the microscope (LEICA MZ12), the digital camera (Hitachi CCD) and an optical fibre providing the episcopic coaxial lighting were placed. A frozen sample prepared as described above was taken out of the vial by carefully crashing the vial, then fixing it on a metal support by tissue freezing medium (LEICA Instruments GmbH) and finally stored in liquid nitrogen. Before the observation, the sample was cut and carefully smoothed down to obtain a surface roughness lower than 1 m. The obtained images were numerically stored in the computer and then were analyzed by Image J 1.34 software that allowed the estimation of the equivalent and mean particle sizes. 2.4. Freeze-drying procedures Frozen samples were freeze-dried (sublimation period) with a laboratory pilot freeze-dryer (USIFROID SMH45, France) using the following protocol [14,15]: • vials with frozen samples placed on the pre-cooled shelf at Ts = −40 ◦ C (−60 ◦ C for 10% sucrose solution) in the freezedryer chamber; • vacuum setting inside the freeze-dryer up to Pc = 10 Pa; • increase the shelf temperature from −40 to −20 ◦ C (from −60 to −40 ◦ C for sucrose solution) at +1 ◦ C/min; • maintain steady freeze-drying conditions all along the primary drying period. A set of 30 vials was put in the same place of the plate of the freeze-dryer for each freeze-drying experiment, and two or three runs were repeated for each experimental condition. In order to determine the drying curves, samples were taken out at the fixed durations with a thief door without breaking vacuum in the chamber, and the weight loss was measured, from which the drying curves were calculated. In order to determine the primary drying rates, the freeze-drying run was stopped after 3–4 h of sublimation, their duration corresponding to about 30–40 wt.% of total ice sublimed.
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3. Results and discussions 3.1. Nucleation temperatures It is well known that supercooled solutions with any control system start their nucleation quite randomly so that, for the same cooling rate and the same experimental conditions, the corresponding nucleation temperatures are largely distributed. From our experiment, the nucleation temperatures were experimentally determined from the temperature profiles recording data, at the minimum point of the undercooling curve when the temperature increases again (due to exothermic nuclei formation) up to the equilibrium freezing temperature. Thus, Fig. 3 shows the frequency distribution of the experimental spontaneous nucleation temperatures for the mannitol, BSA and sucrose systems frozen at the same cooling rate equal to −1 ◦ C/min. These data showed that the nucleation temperatures varied from −2 to −15 ◦ C for all the systems with averaged values equal to −8 ◦ C. All these data were obtained in similar rigorously controlled cooling conditions (cooling rates) and with vials (type, size, cleanness, etc.) as clean and identical as possible. The nucleation of the sample was triggered at selected temperature levels lower than equilibrium freezing temperature. Fig. 4 illustrates the freezing curves obtained with 10% mannitol solutions nucleated by the previously described ultrasound system. When the nucleation process was triggered at low temperature levels corresponding to high undercooling levels, the solution solidified immediately. However, if the nucleation processes was triggered at lower undercooling levels, for example above −3.5 ◦ C, the system was first transformed into a kind of slurry and then reached more gradually the frozen state. First, we confirmed experimentally that the investigated experimented sonication system was useful to trigger the nucleation process of the supercooled solution contained in the vial with a high degree of repeatability. However, it was sometimes observed that some
Fig. 3. Frequency distributions of spontaneous nucleation temperatures.
Fig. 4. Freezing curves of ultrasounds nucleated samples (10% mannitol).
vials did not respond at all to the ultrasound pulse. After more precise observation, it approved that these vials were not placed in stable position on the plates due to the roughness of their bottom or for other unknown reasons, so that the ultrasound wave could not efficiently propagate throughout the whole volume of the vial. Furthermore, in some cases, we also experimentally observed that some samples without triggered nucleation could be nucleated by holding them tightly on the plate. So, it should be mentioned that good mechanical and thermal contact between the vial bottoms, which are quite curved, and the plate surface of the freeze-dryer must be achieved for implementing properly the present system. 3.2. Characterization of ice crystals structure Ice crystal morphologies of 10% mannitol frozen solutions were directly observed by the optical microscopy method in the cold chamber (−25 ◦ C) presented above. The surfaces observed by microscopy were vertical and horizontal sample cuts that corresponded to a cross section of a cylindrical sample frozen in vials. Some typical images that represented the more characteristic trends of each sample are selected and shown in Fig. 5 (vertical cross sections) and in Fig. 6 (horizontal cross sections). In these images, the ice crystals appear in white color and the cryoconcentrated phase, principally corresponding to mannitol crystals, in black colour. One can see in these figures obvious morphological differences of ice crystals structure depending on their nucleation temperatures. Large and directional ice crystals (dendrite type) were confirmed in the sample nucleated at higher temperatures i.e. with lower supercooling degrees, while small and numerous heterogeneous ice crystals appeared in the sample nucleated at lower temperature i.e. with higher supercooling degree. Ice crystal growth in a vial generally starts from the bottom of the vial and progresses to the top, while a highly cryoconcentrated solution layer is formed at the top of the sam-
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Fig. 5. Ice crystal morphologies on vertical cross sections (10% mannitol): (A) upper position, nucleated at −2.04 ◦ C; (B) lower position, nucleated at −2.04 ◦ C; (C) upper position, nucleated at −8.17 ◦ C; (D) lower position, nucleated at −8.17 ◦ C.
ple. It is also confirmed on our vertical sample images (Fig. 5A and C) that the ice crystal initiation by the ultrasound waves occurred at the bottom of the vial, because smaller ice crystals were observed at the vial bottom than at the vial top. This fact was also confirmed by the comparison of ice crystal morphology images between a sample nucleated by ultrasound and a sample spontaneously nucleated at the same temperature. This means that the ice crystal growth nucleated by ultrasound progressed in
the same manner as in case of spontaneous nucleation. Thus, the average crystal size had almost the same value for the two types of nucleation as shown in Fig. 7. It is worth noting that there were no significant differences in the ice morphology of frozen sample prepared with or without ultrasound if they nucleated at the same temperature. Thus, one can say that the ice morphological parameters are mostly determined by the nucleation temperature values for the same operating conditions (cooling
Fig. 6. Ice crystal morphologies on horizontal cross sections (10% mannitol): (A) edge position, nucleated at −2.04 ◦ C; (B) center position, nucleated at −2.04 ◦ C; (C) edge position, nucleated at −7.39 ◦ C; (D) center position, nucleated at −7.39 ◦ C.
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Fig. 7. Ice crystal morphologies (10% mannitol): (1) spontaneous nucleation at −7.64 ◦ C; (2) controlled nucleation by ultrasound at −7.39 ◦ C. Table 1 Mean ice crystal equivalent diameters Nucleation temperature (◦ C)
Upper position (m)
Lower position (m)
Mean value (m)
−2.04 −7.39
142 57.3
103 50.5
122 54.1
rate, sample geometry, etc.). Large and dendrite type ice crystals were also observed in the horizontal cuts of the sample nucleated at higher temperature (Fig. 6A and B); however, they did not always correspond to directional structures like for images of vertical cuts. One can suppose that the three-dimensional image results from the aggregations of dendrite ice crystal in vertical direction. On the other hand, on the horizontal cuts of the sample nucleated at lower temperature, small and numerous ice crystals can be observed (Fig. 6C and D); furthermore, from the images of both vertical and horizontal cuts of the samples (Fig. 5C and D and 6C and D) we could not observe any regular directional ice crystal growth. Thus, it would be reasonable to imagine the three-dimensional structure of the sample nucleated at lower temperature as a network of irregular layers of approximately spherical ice crystals. Ice crystal size distributions calculated by image analysis are plotted on Fig. 8. Three different horizontal images were analyzed for determining each crystal size distribution. The average ice crystal size values are listed on Table 1. One can see that the average crystal size of the sample nucleated at −2.04 ◦ C is more than twice as large as the one resulting from nucleation at −7.39 ◦ C. It was observed from this figure that the ice crystal size distributions also depended on axial positions inside the vial;
larger ice crystals were observed at the upper positions, while smaller ice crystals were observed at the vial bottom. Furthermore, this tendency was confirmed very clearly with sample nucleated at higher temperature. This behaviour suggests that one could not always assume homogeneity of dried layer permeability in considering mass transfer during the drying steps of the freeze-drying process. Besides, from the freeze-drying tests using microbalance technique, Pikal et al. reported that the water vapour mass transfer resistance of the dried product layer was not directly proportional to its thickness [16]. 3.3. Primary drying rate during freeze-drying As shown in the previous sections, we could prepare frozen samples nucleated at selected temperatures, and we could confirm obvious dependency of the ice crystal morphology on their nucleation temperature. Those morphological modifications were expected to modify the mass transfer permeability of water vapour and contribute to accelerate primary drying rates during freeze-drying. Fig. 9 shows the ratio of water mass remained in the sample during primary drying step to the initial mass as a function of sublimation time. We compare in this figure the drying kinetics of a series of frozen samples nucleated, respectively, at around −2 and −8 ◦ C. The sublimation of the samples nucleated at lower temperature progressed at almost the same sublimation rate. Nevertheless, one can observe that the sublimation of the samples nucleated at higher temperature progressed in different manner. At the beginning of the sublimation, the sublimation rates were similar to the rates observed with samples nucleated at lower temperature; however, after two hours of sublimation, the sublimation rate slightly increased, but after five hours of sublimation, it gradually decreased. It is quite
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Fig. 8. Ice crystal size distributions (10% mannitol): (A) nucleated at −2.04 ◦ C; (B) nucleated at −7.39 ◦ C.
evident that these sublimation kinetics are directly related to the ice phase morphology, i.e. to the permeability of the dried layer. The overall primary drying rate values determined gravimetrically are plotted in Figs. 10–12 as a function of nucleation temperature. These plots show proportional relationship between the primary drying rate and the nucleation temperature for each data set, that is to say, the sublimation rate increases with the nucleation temperature. This is a coherent result in accordance with the image analysis data on the ice crystal morphology presented in the previous paragraph. As shown in Fig. 10, at a given temperature, the primary drying rate is approximately the same in presence and in absence of ultrasound. However, ultrasound allows for nucleation at much higher temperature to happen, which yields an increase in primary drying rate (60% e.g. between nucleation temperatures −8 and −2 ◦ C). In case of 10% sucrose freeze-drying (Fig. 12), the enhancement on sublimation rate was much larger: the sublimation rate as doubled by increasing the nucleation temperatures from −8 to −2 ◦ C. Nevertheless,
Fig. 9. Drying curves during freeze-drying (10% mannitol; Ts = −20 ◦ C; Pc = 10 Pa).
for sucrose we set primary drying shelf temperature lower than for the 10% mannitol formulation freeze-drying (i.e. −20 ◦ C for 10% mannitol instead of −40 ◦ C for 10% sucrose freeze-drying) in order to avoid collapse phenomenon. Due to the lower values of water vapour transfer potential during sublimation, the mass transfer was much more controlled by the structural parameters of the dried layers. In case of 5% BSA freeze-drying (Fig. 11), the average sublimation rates were higher than with other formulations, and, the enhancement of the sublimation rate was smaller. However, in this last case, we could also observe a clear relationship between the sublimation rate and the corresponding nucleation temperature. It is noteworthy that spontaneously nucleated samples were located on the same line as the controlled nucleation samples. This behaviour seems to indicate that the ice crystal growth mechanisms are the same for samples nucleated by ultrasound and the samples nucleated spontaneously. It is suggested that,
Fig. 10. Primary drying rates versus nucleation temperatures (10% mannitol). Open symbol: controlled nucleation by ultrasound; closed symbol: spontaneous nucleation.
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4. Conclusions
Fig. 11. Primary drying rates versus nucleation temperatures (5% BSA). Open symbol: controlled nucleation by ultrasound; closed symbol: spontaneous nucleation.
for a given system, the structural parameters of a frozen formulation are strongly dependent on the nucleation temperatures, so that this parameter is relevant for the permeability of dried layer during sublimation step. Thus, these data confirm the adequacy of an ultrasound system to control the nucleation processes and, by the way, to accelerate notably the primary sublimation rates during the freeze-drying process of pharmaceuticals in vials, improving the quality factors related to the structural properties (rehydration facility, water vapour permeability, etc.) and reducing the operating costs related to drying times.
An ultrasound system was set up and was investigated in order to optimize an industrial freeze-drying process in vials by controlling the freezing step. It was clearly observed that the ultrasound system could efficiently trigger the nucleation process of the three kinds of standard formulations investigated in a vial configuration largely used for pharmaceutical proteins freeze-drying in industry. The nucleation temperature of the samples could be controlled at selected values below the equilibrium freezing temperature, corresponding to different undercooling states for the ice crystals growth. Analysis of the ice structure morphology by direct optical microscopy in a cold chamber indicated that nucleation temperatures correspond to different undercooling states for ice crystals growth, that is to say: small and numerous ice crystals are obtained at lower nucleation temperature (higher supercooling degree), while large and directional ice crystals (dendrite type) are obtained at higher nucleation temperature (lower supercooling degree). These morphological modifications on ice crystals clearly impacted on water vapour permeability during the subsequent sublimation step. Consequently, it was proved that a strong correlation exists between the sublimation kinetics and the ice morphology. A significant correlation between the overall primary drying rates and the nucleation temperatures were confirmed (i.e. the drying rates increased with the nucleation temperatures), and, as a result, a great enhancement of their sublimation rates was achieved by increasing their nucleation temperatures from the average spontaneous nucleation temperature to around −2 ◦ C with ultrasound control. Furthermore, the ice crystal size distribution was also depending on axial position inside the vial as more larger ice crystals were observed in the upper part than at the vial bottom; thus, it was suggested that we might have to take into consideration heterogeneous dried layers for a better understanding of mass and heat transfer phenomena during sublimation step of freeze-drying process. Nevertheless, this intra-vial heterogeneity could be largely reduced by using annealing treatments as already found out in our laboratory [17]. Finally, it is expected that this ultrasound control system, after further studies, could be developed and applied for the optimization of pharmaceuticals freeze-drying cycles in industrial condition. Acknowledgements This work is supported by a grant from Japan Society for the Promotion of Science. References
Fig. 12. Primary drying rates versus nucleation temperatures (10% sucrose). Open symbol: controlled nucleation by ultrasound; closed symbol: spontaneous nucleation.
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