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The authors discuss advanced sonocrystallization particle-engineering techniques for manufacturing mesoscopic particles.
The control of the crystal particle size of an active pharmaceutical ingredient (API) is necessary when the final product's performance depends on well defined and, ideally, engineered mesoscopic particles. The size of the particle and the crystal form of the API can influence behavior such as dissolution rate in a biological system. This influence is of particular importance because more than 40% of new molecules being developed are poorly water soluble.
The technology available for particle engineering to improve therapeutic performance has grown rapidly in popularity and sophistication and has created a need for technology that can tailor particles for various applications. The need for improved and efficient drug delivery systems for many new drugs has become apparent, especially when the drugs are administered in a particulate form (1).
In respiratory drug delivery, effective deep-lung deposition is achieved with 1–5-μm particles. Pulmonary administration of various drug substances, particularly small molecules, whether for systemic or local delivery, is especially appealing because of the thin alveolar epithelium wall's large surface area and absorption ability. Local delivery of therapeutics to the lung is an appropriate method for treating conditions such as asthma, chronic obstructive pulmonary disease (COPD), infections, and disease states such as cystic fibrosis. Other important therapeutics areas that pulmonary delivery can address include those where a rapid onset of action is required (e.g., diabetes, neuropathic pain, migraine), and systemic action of peptides, proteins, and oncolytics.
Top-down and bottom-up methods
Size reduction, or "top-down," destructive methods for preparing mesoscopic particles such as fluid-energy milling, also known as micronization (including jet milling and fluidized-bed jet mills), achieve the target size range, but the high energy required for such processes often damages the crystal's surface. This damage leads to highly charged and cohesive particles, which result in the chemical and physical instability of the drug. A significant proportion of micronized particles may be too fine and of an unsuitable shape for a given use. These qualities lead to undesirable surface polymorphological transformation, which may form amorphous structures that are undesirable for applications such as inhaled treatments. In addition, the particle properties can vary from batch to batch, thus causing problems in downstream processing and product formulation. Micronization can also generate considerable heat, which may be incompatible with the material of interest (e.g., a low-melting solid API).
Constructive "bottom-up" or "molecule-to-particle" techniques provide a way to avoid these destructive techniques and appear to offer superior alternative production methods that yield better products. The US Food and Drug Administration would theoretically favor constructive techniques because they are consistent with its quality by design initiative aimed at improving the invention, development, and commercialization of structured products using technologies that provide superior product quality, per the ICH Q8 guidelines of 2004.
One such technology, sonocrystallization, uses the excellent dispersive and crystal-nucleation properties of transient cavitation to produce microcrystalline particles when a drug–solute solution makes contact with an antisolvent. The literature offers a review of particle-engineering technologies (2). Sonocrystallization can also be applied to the preparation of aqueous nanosuspensions or submicrometer colloidal dispersions of a pure drug. These dispersions are ideal formulations to improve bioavailability (3, 4).
Nanosizing reduces the size of the API to the submicrometer range (typically 100–200 nm) in an aqueous media. Surfactants or polymers stabilize the API concurrently (5). Nanosuspensions can be dried using conventional techniques such as spray drying or lyophilization.
Prosonix (Oxford, UK) has developed advanced sonocrystallization particle-engineering techniques for manufacturing mesoscopic particles. This article discusses these techniques, including the proprietary Solution Atomization and Crystallization by Sonication (SAX) and Dispersive Crystallization with Ultrasound (DISCUS) methods. The DISCUS method involves recirculation and miscible and immiscible (emulsion-oriented) systems. The article also reviews the factors that affect the production of tailor-made particles suitable for efficient delivery and improved therapeutic performance.
The power of ultrasound
Sonocrystallization, or ultrasound-assisted crystallization, relies on the power-ultrasound (20–100 kHz) and extended-sonochemistry (100 kHz–2 MHz) bands of the acoustic-frequency range. By comparison, human hearing responds to frequencies between 20 Hz and 19 kHz.
Ultrasound involves mechanical vibrations that transfer energy by molecular motion. One consequence of ultrasound vibration is cavitation, the transient high-energy microbubbles formed when power ultrasound is applied to a liquid medium. The energy release associated with this phenomenon, once dissipated, can lead to permanent changes in the molecular structure of entities in close proximity.
Conversely, ultrasound can be used in various medical applications, diagnostic pulse-echo techniques, attenuation measurements, and particle separation, without any change to the medium or species suspended or dissolved in the liquid medium.
The acoustic bubble
The advantages that sonocrystallization offers over conventional crystallization largely result from cavitation. The microbubbles caused by cavitation are short-lived microreactors where responses and reactions such as chemical reaction and crystal nucleation take place in a uniquely favorable environment as large amounts of energy are transferred to reagent molecules in an extremely short time (6, 7).
Most ultrasound work has used either intense-probe or ultrasonic-bath-based equipment. Although cavitation is the essential element for successful sonocrystallization, including ultrasonic wet milling, it can be harmful to an ultrasound-radiating surface. Cavitation usually damages the ultrasonic device and creates problems with ultrasonic probes, where cavitation occurs close to the radiating surface. These engineering limitations can be overcome, however.
Small particles and sonocrystallization
Sonocrystallization can be applied at any stage of pharmaceutical manufacturing and lends itself to polymorphic systems (7, 8). The technique uses transient acoustic cavitation to assist in the nucleation of metastable solutions. By controlling nucleation, one controls and improves crystal-size distribution, morphology, impurities, polymorphism, and solid–liquid separation. Ultrasound can also induce secondary nucleation by mechanically disrupting crystals or loosely bound agglomerates.
Companies such as Prosonix, GlaxoSmithKline (Brentford, UK), AstraZeneca (London), and Syngenta (Basel) have patented crystallization methods based on ultrasound-assisted precipitation, principally for the preparation of nano- and microcrystalline particles (9–13). The cavitation-induced effects can be so strong that nanosuspensions can be prepared when the antisolvent is water.
Molecule-to-particle techniques using power ultrasound such as antisolvent precipitation take advantage of the excellent dispersive and crystal-nucleation properties of transient cavitation. Several companies that manufacture microparticles and nanosuspensions are adopting these techniques. Precipitation conditions must be chosen to maximize crystal nucleation at the expense of growth, which will necessitate having the appropriate prevailing supersaturation.
Rabinow gives a useful overview of the preparation, delivery, and performance of nanosuspenions, but in simple terms, a solution of the API must be added to the antisolvent (water) at an optimal rate to generate micrometer- and, where necessary, submicrometer-sized particles (3). One problem with this strategy is that supersaturation can force a phase separation and lead to oiling or rapid precipitation of amorphous forms and metastable polymorphs, because of slow nucleation kinetics. Yet more stable solid forms can eventually form, as indicated by Ostwald's law of stages. How does power ultrasound help?
Acoustic cavitation can have remarkable benefits for the system of interest, whether it is the manufacture of microparticles or nanosuspensions. Cavitation not only improves mixing and increases the diffusion of molecules toward a precrystal cluster or nucleus, it facilitates the formation of a crystalline phase (14). This latter effect is attributed to dramatic temperature and pressure changes, shockwaves, and rapid local cooling rates (15).
The continuous mixing of a solution of API in a suitable miscible solvent with an antisolvent, whether water or an organic solvent, in a flow cell fitted with an ultrasonic probe shows the potential of the sonoprecipitation and sonocrytallization of microcrystalline pharmaceutical products (11). During the course of the process, the solvent–antisolvent ratio always remains constant, and flow rates must be balanced. Nevertheless, this continuous technique has potential advantages.
Water-insoluble drug substances can be dissolved in a water-miscible organic solvent that can then be added to water in the presence of an ultrasonic field. The ultrasound can be applied using an immersed ultrasonic probe to generate a dispersion. The form of the dispersion depends upon ultrasonic energy and the presence or absence of stabilizers (12). Even though acoustic cavitation occurs during the initial mixing, amorphous particles are produced. Amorphous particles are considered essential for avoiding crystal growth if crystalline phases are present. Applying continuous ultrasound after mixing leads to the formation of crystalline submicrometer-sized particles through a solution-mediated amorphous-to-crystalline transition.
If it is important to avoid organic solvents and the organic compound of interest has a suitably low melting point, sonocrystallization can aid antisolvent crystallization in several ways. Sonocrystallization typically helps through a process that disperses the melted liquid into the antisolvent water, solidifies the melted droplets, and subsequently or concurrently crystallizes the compound (13). The ability to apply any particle-engineering methodology or technology on an industrial scale is paramount.
Dispersions, sonocrystallization, and DISCUS
Antisolvent precipitation is an indispensable tool for the process chemist. Crystallization can often be achieved by mixing a solution of the drug substance with an antisolvent so that, after mixing, the solution is supersaturated and crystallization occurs. The extremes of supersaturation usually cause precipitation of amorphous and ultrafine particles.
Figure 1 (ALL IMAGES ARE COURTESY OF PROSONIX.)
One can take advantage of these effects by mixing in the presence of an ultrasonic field. The two streams can be mixed either in continuous mode, using Prosonix's SonoLab ultrasonic devices, or in a recirculation process loop (see Figures 1 and 2). In the latter, the antisolvent stream is recirculated rapidly through the flow cell while the optional feed API solution is fed slowly into the flow cell (see Figure 2). Flow-rate ratios in this process often exceed 100:1 in favor of the recirculating antisolvent. High flow rates lead to rapid dispersion and crystallization of micrometer- and submicrometer-sized particles, which can then be isolated by spray drying, for example. Chemists would most often add antisolvent (e.g., the optional feed in Figure 2) to the API solution, but this reverse-antisolvent process is essential to avoid the particle growth that occurs during a normal antisolvent process. Prosonix's DISCUS is a dispersive technology that uses ultrasound.
Figure 2 (ALL IMAGES ARE COURTESY OF PROSONIX.)
Prosonix has produced several microcrystalline steroids using this reverse process, which is an effective means of preparing mesoscopic crystalline particles at industrial scale. The reverse process provides many options. For example, the nonsolvent–solvent system may be miscible (e.g., an ethanol solution dispersed into heptane) or immiscible (e.g., dichloromethane or toluene dispersed into water). Volatile solvents or solvent azeotropes can be continuously removed. One can also feed a melt of the API (provided its melting point is not exceptionally high) into the recirculating antisolvent. All these methods can be used for preparing aqueous nanosuspenions, often with the use of stabilizers.
Ultrasound-mediated emulsion crystallization is a novel particle-engineering technique to facilitate the formation of submicrometer- to micrometer-sized particles for improving therapeutic efficiency. This technique is beneficial for poorly water-soluble drug candidates.
In a typical process, a drug is dissolved in an organic solvent, which is immiscible with the nonsolvent of choice. Ultrasound is applied to achieve a stable emulsion. Each emulsified droplet can be subjected to heat or mass-transfer effects to achieve evaporation, cooling, or diffusion and bring about the required degree of supersaturation and crystal nucleation. The application of ultrasound assists in the dispersion and stabilization of the drug particles in the nonsolvent.
The mechanism of particle formation can be seen as a sequential process governed by applied ultrasound. After emulsification, the droplet size of the organic solution decreases because the organic solvent evaporates at high temperatures. Concurrently, the drug concentration within the droplet increases with time. When the supersaturation of drugs in the shrinking droplet is high enough, the drug, and sometimes the excipient, crystallizes.
Two mechanisms allow the crystal nuclei to form larger drug particles: the drug molecules in the organic phase diffuse toward the nuclei and condense, and the nuclei collide and coagulate together to form larger particles (16). At this stage, applied ultrasound plays a significant role in controlling the size of resulting particles. Although the particles can grow, predominantly by coagulation, ultrasound helps to deagglomerate and form submicrometer-sized crystalline particles. At high supersaturation, or a low particle-growth rate, submicrometer-sized particles can be produced. Because the concentration of the organic solvent in nonsolvent is extremely low, the particle growth through Ostwald ripening may be expected to be low relative to precipitation (17).
In all these dispersive crystallization methods, many parameters can influence particle characteristics and require optimization. They include ultrasonic power, concentration and feed rate of API solution, and temperature and flow rate of antisolvent. For emulsion crystallization, particle size is also governed by the interplay between dispersion, coagulation, deagglomeration, supersaturation, and Ostwald ripening (16, 17). The suspensions may be dried by common means such as supercritical carbon-dioxide extraction, spray drying, lyophilization, and centrifugation. Small-scale automated equipment can be used for designed studies to facilitate scale-up to the pilot plant.
Aerosolization, sonocrystallization, and SAX
Particle-engineering technology, especially techniques that control the production of microcrystalline particles with a narrow size distribution and overcome the disadvantages of common micronization techniques, has shown great promise in preparing particulate pharmaceuticals with defined physicochemical properties.
Techniques for producing drug particles include spray-drying, which involves generating an aerosol of droplets from a solution of the drug and subsequent drying of the droplets to solidify the particles. Spray-drying is one of the most widely used industrial processes involving particle formation and drying. It is highly suited for the continuous production of dry solids in either powder, granulate, or agglomerate form from, for example, liquid feedstocks such as solutions, emulsions, and suspensions. The end product of the spray-drying process should comply with quality standards for parameters such as particle-size distribution, residual moisture content, bulk density, and particle shape. A disadvantage of conventional spray-drying techniques is that the particles produced tend to be amorphous—perhaps as much as 100%—rather than in a crystalline particulate form because solidification is typically rapid.
Newer techniques offer greater control of crystallinity and morphology. To achieve optimal drug delivery to the lung, it is important to ensure that the drug is formulated into particles of the appropriate aerodynamic size, shape, and density.
Prosonix developed the SAX technology for preparing micro- and nanocrystalline particles for drug delivery and now seeks to industrialize the process. These particles are used principally for inhaled medicines for asthma and COPD as well as antibiotics (18). SAX and other sonocrystallization technologies could change the manufacturing of inhaled medicines significantly.
Multiple therapeutic areas
SAX is a scalable, economic technology that generally functions at ambient temperature and pressure (19–21). This solution-to-particle methodology avoids many of the problems apparent with the various methods available to date. Importantly, SAX also allows the production of spherical drug particles with unique nanotopology and superior aerodynamic properties. These characteristics improve the sorption characteristics by virtue of the increased particle surface area for a given size and volume.
SAX has been applied to many compounds to date, including new chemical entities (NCEs) and many steroidal compounds for asthma, COPD, and topical creams and gels. SAX has been used to prepare budesonide, fluticasone propionate, beclamethasone dipropionate, ciclesonide, betamethasone and mometasone furoate, -agonists for asthma and COPD, salmeterol xinafoate, formoterol fumarate, and salbutamol. Prosonix has also used SAX successfully for producing aminoglycoside and cephalosporin antibiotics.
SAX provides the platform for a particle-engineering solution whereby a single droplet containing the two APIs in an exact ratio can be converted to a combination particle that contains the same drug substances as separate crystalline entities. In combination therapies for asthma and COPD, where particle engineering is essential to formulation, the APIs often have synergistic action at the molecular and cellular levels (e.g., inhaled steroids and long-acting agonists) and must be delivered in an exact ratio (22).
The particles of both ingredients should have a high degree of crystallinity. Importantly, they must arrive at the site of action in the lung together. Triple therapy, using an anticholinergic as the third component, is possible with SAX combination-particle technology. The preparation of such combination particles could also have a significant part to play in areas of cancer chemotherapy, where multiple drugs are prescribed.
The principles of SAX are simple (see Figure 3). SAX involves the formation of a drug-substance solution followed by its atomization, controlled evaporation of the solvent, collection of the preconcentrated viscous droplets in a vessel containing nonsolvent, and crystallization through nucleation with power ultrasound. The product slurry is then transferred to solid isolation, preferably by spray-drying or supercritical carbon-dioxide drying.
Figure 3 (ALL IMAGES ARE COURTESY OF PROSONIX.)
SAX has developed considerably since its inception and is now available for full-scale demonstration, proof-of-concept studies, and establishing the proof of process for scale-up studies. The technique provides opportunities to evaluate engineered particles for their superior performance in terms of therapeutic efficiency and long-term stability. SAX technology could potentially be the platform to improve the manufacture of superior particles for existing drug moieties, substances, and NCEs in development. Figure 3 shows photographs of the SAX technology for research and development.
Solvent and solubility
Many parameters can influence particle characteristics and require optimization. The concentration of the solute in the solution has considerable influence on the particle size and the volume of the drug particulate. The particle size and the volume tend to increase as the concentration of solute in solution increases. The main reason for this is the increase in the relative abundance of solute in the atomized solution compared to that of a dilute solution. The solute concentration should be optimized by considering the desired particle size and the process economics. As a rule of thumb, the drug that is selected for the process should exhibit optimum solubility in the selected solvent. The interaction of the drug with the selected solvent or nonsolvent should be given due consideration to discover the possibility of solvate generation, which may be undesirable in certain cases.
Atomizer and atomization
The choice of atomizer and its operation govern the particle size of the drug. The atomizer employed in the process should efficiently atomize the drug solution to microdroplets so that they evaporate and contract to yield microcrystalline particles. Good control of gas pressure can help in the size distribution of resulting particles (see Figure 4). The increase in gas pressure can lead to smaller droplet size, which in turn yields a smaller particle following evaporation and concentration of the droplet. Submicrometer-sized particles are routinely obtained through careful selection of pressure conditions employed with the desired atomizer. The two-fluid nozzle performs better and is ideally suited to scale-up. It is important to optimize the gas pressure and flow rate to achieve the best atomization and evaporation characteristics.
Figure 4 (ALL IMAGES ARE COURTESY OF PROSONIX.)
Depending on the choice of solvent, the gas pressure and flow rates are adjusted to achieve a uniform temperature profile along the length of the column. For a given compound and ideal choice of dissolution solvent, it is imperative to achieve an ideal and almost precise temperature profile along the length of column at a given solution feed rate and corresponding gas pressure.
Productivity and flow rate
Particle size can be fine-tuned by varying the feed-solution flow rate. The particle size can be decreased by slowing down the feed-solution flow rate at a given solute concentration. Similarly, the particle size can increased by speeding up the feed-solution flow rate. Although the particle size can be tuned by adjusting the feed-solution rate, the corresponding gas pressure has a significant effect on the resulting particles (see Figure 5). This feature is important in using SAX for morphology control.
Figure 5 (ALL IMAGES ARE COURTESY OF PROSONIX.)
Temperature plays a major role in the SAX technology. It mainly influences the evaporation rate of the solvent employed in the process. Ultimately, it governs the particle size of the drug. Although particle size is the key parameter affected by temperature, thermal effects can influence the amorphous or crystalline character of the particle. This idea is of paramount importance when nonsolvent temperature is selected.
Ultrasound and nonsolvent
Power ultrasound and the selection of the appropriate nonsolvent are the keys to collecting concentrated droplets and inducing ultrasound-assisted nucleation. Applying ultrasound delivers astonishing results in terms of particle shape (see Figure 6). However, the choice of solvent has an equal effect on the resulting particles. SAX technology in general has shown potential in morphology control. It helps to generate the targeted particles and engineer them to the requirements of their ultimate end use.
Figure 6 (ALL IMAGES ARE COURTESY OF PROSONIX.)
Useful scale-up equipment for ultrasonic processing has become available during the past 15 years. Probe-based systems generally have inherent problems associated with cavitational damage and inefficient power consumption. Adapting such systems for the pharmaceutical industry is difficult because they must be approved for use with flammable solvents—an essential feature of equipment for manufacturing fine chemicals and APIs.
Prosonix has designed its piezoelectric-based Prosonitron equipment to allow focused distribution of acoustic energy into a liquid by using several low-power transducers (21 in a 5-L flow cell) bonded to the outside of a cylindrical duct, as shown in Figure 1, (7, 8, 23). A new cells with one row of seven transducers, (see Figure 2) was designed specifically for kilo-laboratory and pilot-plant use, either in recirculation or continuous mode.
The key feature of the Prosonitron design is that the most significant transient cavitation takes place in the center of the device. The equipment is modular and easily cleaned for production according to current good manufacturing practice. It also can be steam-sterilized and is therefore useful for the aseptic manufacture of antibiotics, for example.
For manufacturing operations, it is more usual to configure a small ultrasonic flow cell into a recirculation loop, as shown in Figures 1 and 2, with a larger and existing reactor, crystallizer, bioreactor, or fermenter.
It seems more sensible to use this retrofit option and to be able to wheel the ultrasonic module from vessel to vessel or plant to plant than to build larger ultrasonic reactors. This configuration can also be modified easily to incorporate a secondary feed stream of solute solution or antisolvent directly into the intense ultrasonic field. For techniques such as SAX, the Prosonitron is a key element in large-scale production equipment (see the left of Figure 3).
With an ever-increasing demand for mesoscopic particles of pharmaceutical materials, whether microcrystalline or nanocrystalline colloidal in form, with defined physical and chemical characteristics, the benefits offered by ultrasound-assisted particle-engineering technologies are valuable. Because ultrasound-assisted technologies are single-stage, solution-to-particle processes and have the potential to meet the scale-up requirements, attractive solutions are available to address many of the problems in engineering pharmaceutical materials and particle design.
Ultrasound should have a bright future in industrial processing, crystallization, and particle engineering. True industrial adaptation of the techniques discussed will necessitate the design and building of new ultrasonication equipment for laboratory use and production in a safe, effective, and economic manner.
Many processing opportunities can be approached with flow-cell technology. Power ultrasound equipment should find its way into many manufacturing facilities. New techniques such as SAX and DISCUS could become the default methods for producing perfect mesoscopic particles.
Graham Ruecroft* is chief technical officer, and Dipesh Parikh is a senior scientist at Prosonix, The Magdelen Centre, Robert Robinson Ave., Oxford Science Park, Oxford OX4 4GA, UK, tel. +44 1865 784244, firstname.lastname@example.org
*To whom all correspondence should be addressed.
1. B. Aungst, "Intestinal Permeation Enhancers," J. Pharm. Sci. 89 (4), 429–442 (2000).
2. A.H.L. Chow et al., "Particle Engineering for Pulmonary Drug Delivery," Pharm. Res. 24 (3), 411–437 (2007)
3. B.E. Rabinow, "Nanosuspensions in Drug Delivery," Nat. Rev. Drug Discov. 3 (9), 785 (2004).
4. C. Leuner, J. Dressman, "Improving Drug Solubility for Oral Delivery Using Solid Dispersions," Eur. J. Pharm. Biopharm. 50 (1), 47–60 (2000).
5. Y. Wu, F. Kesisoglou, S. Panmai, "Nanosizing—Oral Formulation Development and Biopharmaceutical Evaluation," Adv. Drug Deliv. Rev. 59 (7), 631–644 (2007).
6. Synthetic Organic Sonochemistry, J.L. Luche, Ed. (Plenum Press, New York, 1998).
7. G. Ruecroft et al., "Sonocrystallization: The Use of Ultrasound for Improved Industrial Crystallization," Org. Process Res. Dev. 9 (6), 923–932 (2005).
8. G. Ruecroft, "Sound Science in Molecules and Particles," Speciality Chemicals Magazine (June), 60 (2008).
9. Prosonix. Entrainment in Anti-Solvent. Br. Patent Application 07.05159.2, 2007.
10. Prosonix, Crystalline Particles Using Immiscible Anti-Solvent: Solvent, Br. Patent Application 07.11680.9, 2007.
11. Glaxo Group Limited, Novel Process, Patent WO/2003/035035, 2003.
12. AstraZeneca, Process for the Preparation of Crystalline Nano-Particle Dispersions, Patent WO/2004/009057, 2004.
13. Syngenta Limited, Process for Preparing a Crystal Suspension, US Patent 6,517,853, 2003.
14. Z. Guo et al., "Effect of Ultrasound on Anti-Solvent Crystallization Process," J. Cryst. Growth 273 (3–4), 555–563 (2004).
15. C. Virone et al., "Primary Nucleation Induced by Ultrasonic Cavitation," J. Cryst. Growth 294 (1), 9–15 (2006).
16. X. Chen et al., "Rapid Dissolution of High-Potency Danazol Particles Produced by Evaporative Precipitation into Aqueous Solution," J. Pharm. Sci. 93 (7), 1867–1878 (2004).
17. X. Chen et al., "Preparation of Cyclosporine A Nanoparticles by Evaporative Precipitation into Aqueous Solution," Int. J. Pharm. 242 (1–2), 3–14 (2002).
18. University of Bath, Process for the Production of Particles, Patent WO/2004/073827, 2004.
19. R. Price, J.S. Kaerger, "Processing of Spherical Crystalline Particles via a Novel Solution Atomization and Crystallization by Sonication (SAXS) Technique," Pharm. Res. 21 (2), 372–381 (2004).
20. G. Ruecroft, "Power Ultrasound and Particle Engineering: Crystals for Drug Delivery and Formulation," Chim. Oggi 25 (3), 12–14 (2007).
21. G. Ruecroft, "Sound, Science, and Crystals: Improved Crystallization and Particle Engineering," Screening 2 34–36 (2007).
22. M. Hannay et al., "Overcoming the Challenges of Developing Combination Products in Different Inhalation Device Platforms," Respiratory Drug Del. 319 (2008).
23. Prosonix Limited, Process and Apparatus for Irradiating Fluids, Patent WO/2000/35579, 2000.