Supercritical Fluid and Subcritical Fluid

SUPERCRITICAL FLUID AND SUBCRITICAL FLUID
Keyur Vasava..
2010-2011

Introduction Pharma science is witnessing a lot of research targeted at meeting delivery and manufacturing demands of the new age molecules. Entirely new technologies and modifications of existing technologies are being developed to address these needs. Supercritical fluid technology (SFT) has been used in many fields for decades, such as the food industry, chemical processing, polymers, textile, forest product industries, and in the cleaning of precision parts other than pharmaceuticals. In the pharmaceutical field it has been widely used for the extraction of natural products like aromatic oils and caffeine, etc. Newer areas of their application have appeared, such as particle size reduction, and designing of novel drug delivery systems.
Important Definitions: 1) Critical Temperature: If the temp. is elevated sufficiently, a value is reached above which it is impossible to liquefy a gas irrespective of the pressure applied. This temp. above which a liquid can no longer exist, is known as critical temperature. 2) Critical Pressure: The pressure required to liquefy a gas at its critical temperature is the critical pressure, which is also the highest vapour pressure that the liquid can have. Basis of SF Technology: The first report which describing a potential process using a supercritical fluid as medium for particles production was published by Hannay and Hogarth: We have then, the phenomenon of a solid with no measurable gaseous pressure, dissolving in a gas. When the solid is precipitated by suddenly reducing the pressure, it is crystalline, and may be brought down as snow in the gas, or on the glass as a frost, but it is always easily redissolved by the gas on increasing the pressure (Hannay and Hogarth, 1879). A fluid reaches the supercritical region when heated and pressurized above its critical pressure and temperature; the critical point represents the end of the vaporization curve in the PT phase diagram (Fig. 1). The supercritical status does not represent a specific aggregation state, but it corresponds to a region where the physico-chemical properties of the material are intermediate between those of the liquid and the gas. The macroscopic appearance of a fluid at Page 1

the critical point is that of a homogeneous and opalescent system without apparent phase separation because, at this point, the density of the gas and liquid are identical. Like a gas the SCF shows lower viscosity and higher diffusivity relative to the liquid. These properties facilitate mass transfer phenomena, such as matrix extraction or impregnation. Like a liquid, the SCF shows a density value high enough for exerting solvation effects. A SCF is dense but highly compressible, thus, any pressure change results in density alteration and, consequently, in solvent power variation (Brunner, 1994). In the vicinity of the critical point, the compressibility is high, and a small pressure change yields a great density modification.
FIG.-1 Phase Diagram of Supercritical region.
Choice of SFs: All gases can form SCF above specific sets of critical conditions (P, T), but it should be kept in mind also that, for many cases, the transition to the supercritical state occurs at high temperatures not compatible with pharmaceutical compounds (e.g., SC water, Table 1). The critical P, T values increase with the molecular weight or intermolecular hydrogen bonding or polarity [18]. Not only mild processing conditions, but also safety and affordable economics are valid criteria to choose the supercritical fluid. For example, Xenon and SF6 (when sufficiently purified) have low critical values, but remain too expensive for commercial use. Gases such as N2O or ethane have low critical values, but can generate explosive mixtures and are therefore unsafe to handle. SCF trifluoromethane (CHF3), which is chemically inert, nonflammable, has low toxicity and a low critical temperature and pressure. Furthermore, CHF3 has a strong Page 2

permanent dipole moment (1.56 D), and can thus solubilize pharmaceutical compounds. However, more than 98% of applications have been developed using carbon dioxide as the SCF because of its low critical temperature (31.18 C) and pressure (7.4 MPa), inexpensiveness, nonflammability, non-toxicity, recyclability and environmental benignity. The field of supercritical fluid is still young and the SCF technologies are not yet widespread throughout the pharmaceutical industry, which is likely due to the high cost of the equipment and the weak knowledge of this area. Tc (.C) 374 16.6 45.5 36.5 25.9 31.3 9.1
MATERIAL H2 O Xenon SF6 N2O CHF3 CO2 C2H4
Pc(MPa) 22 5.9 3.8 4.1 4.7 7.4 5.1
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FIG.-2 Phase Diagram of CO2
PROCESSING USING SUPERCRITICAL FLUID: Supercritical fluids have been proposed for many different unit operations; nevertheless all processes are based on some techniques which can be divided in four groups: Operations where the SCF acts as a solvent (RESS, RESOLV); Operations where the SCF acts as an antisolvent (GAS, PCA, ASES, SEDS); Particles from a gas-saturated solutions (PGSS, DELOS, CPCSP); CO2-assisted spray-drying (CAN-BD, SAA). 1. Operations where the SCF acts as a solvent: A) RESS process (Rapid Expansion of Supercritical Solution) The RESS process (Rapid Expansion of Supercritical Solution) consists of the saturation of the supercritical medium with a solute followed by a rapidly depressurization (expansion) of the solution through a heated nozzle at supersonic speed. The rapid and uniform pressure drop, obtained by passing from supercritical to ambient conditions, leads to a dramatic and instantaneous decrease of solvent power (solute supersaturation) and as a consequence, to a rapid nucleation of the solute in form of very small particles with uniform size. These particles are completely dry, solvent free, and they do not need further processing. The morphology and size distribution of the precipitated material is a function of its pre-expansion concentration and expansion conditions. The pre-expansion concentration is Page 4

dependent on the choice of SF, nature of solute, addition of co-solvents and operating pressure and temperature. The higher the pre-expansion concentration, the smaller the particles and narrower the particle size range.
ADVANTAGES OF RESS: The simple control of process parameters The relatively easy implementation on lab-scale when a single nozzle is used The absence of organic solvents DISADVANTAGES OF RESS: Limited the development of this technique: the difficulty in scaling-up The possible particles aggregation and nozzle blockage The need of large amount of SCF The poor solubility of most pharmaceutical compounds in supercritical CO2.
FIG.-3 RESS Equipment Concept.
B)
RESOLV process RESOLV (Rapid Expansion of a Supercritical Solution into a Liquid Solvent) represents a variation of RESS. This technique was studied in order to minimize the particles aggregation during the jet expansion. Here, the supercritical solution is depressurized through an orifice into a collection chamber containing an aqueous solution at room temperature. Various water-soluble polymers or surfactants are added to the aqueous medium for stabilizing the obtained nanoparticle suspension. Page 5
2. Operations where the SCF acts as an antisolvent: These techniques were proposed to process molecules with poor solubility in SCFs. Here the pressurized CO2 acts as an antisolvent for precipitating a solute from an organic solvent solution. The solute is first dissolved in an organic solvent, and then the pressurized CO2 is put in contact with the solution. The principle is based on (i) the ability of the organic solvent to dissolve a large amount of gas; (ii) the mutual miscibility of the organic and SCF phases; and (iii) the low affinity of the SCF for the solute. The CO2 diffuses into the organic solvent leading to the solvent evaporation into the gas phase; then, the volume expansion determines a density reduction, which lowers the solvent power of the organic solvent which leads to the precipitation of the solute. One of the requirements for this approach is that the carrier solvent and the SF antisolvent must be at least partially miscible. This process works in a semi batch mode, with the supercritical solvent introduced into an already existing stationary bulk liquid phase. This mode offers better control over the particle characteristics as governed by the rate of addition of the SF. However, the liquid phase cannot, in general, be completely removed, and requires additional processing steps before a dry product can be recovered. The effect of processing variables such as temperature, pressure, stirring rate, concentration of the injection solution, rate and temperature of the carrier solution, nature of liquid solvents and choice of the SF on the physical properties of the end product have to be optimized when looking to obtain any product of desired characteristics. Different processes based on the different mixing modes between the organic solution and the SCF were designed. In the Gaseous Antisolvent (GAS) the precipitation vessel is loaded with the solution then, the SCF is introduced into a vessel until the final pressure is reached.
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FIG.-4 GAS equipment concept.
In the Particles by Compressed Antisolvent (PCA) and Supercritical Antisolvent (SAS), the CO2 (supercritical for SAS, or subcritical for PCA) is first pumped inside the high-pressure vessel until the system reaches the fixed pressure and temperature, then, the organic solution is sprayed through a nozzle into the SCF bulk determining the formation of the particles that are collected on a filter at the bottom of the vessel.
FIG.-5 SAS equipment concept
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FIG.-6 ASES equipment concept
The Aerosol Solvent Extraction System (ASES) is similar to the SAS technique except that, in this case, the solution and the antisolvent are simultaneously sprayed into the precipitation vessel.
FIG.-6 SEDS equipment concept
Simultaneous spraying of the solution and the antisolvent occurs in the case of Solution Enhanced Dispersion by Supercritical Fluids (SEDS) as well. Compared with the other antisolvent techniques SEDS is characterized by the use of a co-axial nozzle designed Page 8

with a mixing chamber, which allows the attainment of better mixing by enabling a turbulent flow. SEDS was also successfully used to process aqueous solution of peptides and proteins despite the limited solubility of water in SC-CO2. SEDS overcomes this problem by using a co-axial three-component nozzle, in which the organic solvent, the SCF, and the aqueous solution, as separate streams, meet in the mixing chamber before being introduced in the precipitation vessel. The temperature and pressure together with accurate metering of flow rates of drug solution and SF through a nozzle provide uniform conditions for particle formation. This helps to control the particle size of the product and, by choosing an appropriate liquid solvent, it is possible to manipulate the particle morphology. Although the antisolvent techniques are very promising for particles engineering, the complete solvent removal still represents an issue: a final wash-out treatment with supercritical CO2 through the precipitated particles may be required. 3. Particles from a gas-saturated solutions Particles from gas-saturated solutions (PGSS), in its most interesting application for pharmaceutics, implies the dissolution of the compressed gas (super- or subcritical) into a melted material (usually a polymer) followed by the rapid depressurization of the gas-saturated solution through a nozzle that causes the formation of particles. The interaction between CO2 and the polymer reduces inter chain polymer bonds giving rise to an enhanced polymer segmental mobility that in turn results in a reduction of the glass transition or melting point. The extent of the glass transition or melting point depression depends on the amount of CO2 dissolved in the polymer. In the rubbery or liquid state the polymer can be used as coating agent, to produce foams, or to incorporate drugs, affording a molecular dispersion which can be extruded or sprayed at lower pressure to obtain drug loaded microparticles. Obviously, polymer plasticization can be also induced thermally or by means of organic solvents, however major drawbacks of these approaches are the difficulty to process heat-sensitive materials and the possible presence of residual solvent in the final product. These are points of major concern especially in pharmaceutical, biotechnological and food applications.
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This technique offers numerous advantages: the pressure used is lower than that necessary for RESS; the required amount of gas is relatively small; no organic solvents are required; it can work in continuous mode; the process affords a good yield. On the other hand, the poor control of the particles size and particle size distribution represents an issue that has still to be addressed. Nonetheless the balance between advantages and drawbacks should be considered positive, and this technique might play a prominent role in the future of drug delivery especially for the preparation of microparticulate systems. Several PGSS plants are already running with capacities of some hundred kilograms per hour (Natex, Austria, Thar Technologies, USA, Uhde HPT)
(http://www.natex.at, http://www.thartech.com, http://www.UhdeHPT.com). Based on the principle of PGSS also DELOS and CPCSP processes have been developed. In the Depressurization of an Expanded Liquid Organic Solution (DELOS) the CO2 expands in an autoclave where an organic solution of the solute to be micronized is dispersed. Then the ternary mixture solutesolventcompressed gas is depressurized by rapid reduction of the system pressure to atmospheric conditions in an expansion chamber. During the expansion the mixture cools down: the temperature drop is the driving force that causes the nucleation and precipitation of the drug. In this process the CO2 does not act as an antisolvent, but as a co-solvent to nebulize and cool the organic solution. The process is not necessarily supercritical; in fact the operative pressure does not exceed the critical point of the CO2/solvent mixture. The Continuous Powder Coating Spraying Process (CPCSP) was proposed by for coating powders. In the CPCSP, the main components (binder and hardener) are melted in separated vessels to avoid a premature interaction with the polymer. The molten polymer is fed into a static mixer, and homogenized with compressed carbon dioxide. Then, the different components are intensively mixed and the formed solution expanded through a nozzle into a spray tower.
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FIG.-7 PGSS equipment concept
4. CO2-assisted spray-drying In these aerosolization techniques the supercritical CO2 is used to assist the nebulization of the solution of the compound to be processed. The mechanism of this process is close to classical micronization by spray-drying. The substance is dissolved or suspended in water or ethanol or both, and the solution or suspension is intimately mixed with the SCCO2. The formed emulsion is rapidly decompressed through a suitable device. In the case of CAN-BD (Carbon dioxide Assisted Nebulization with a Bubble Dryer) the near-critical or supercritical CO2 and the solution are pumped through a near zero volume to give rise to an emulsion which expands through a flow restrictor into a drying chamber at atmospheric pressure to generate aerosols of microbubbles and microdroplets that are dried by a flux of warm nitrogen. In the case of SAA (Supercritical Fluid-Assisted Atomization) the supercritical CO2 and the solution are mixed into a vessel loaded with stainless steel perforated saddle which assures a large contact surface between liquid solution and the SCF; then the mixture is sprayed in a precipitator at atmospheric pressure under a flow of hot N2. The main difference between CAN-BD and SAA processes is represented by the mixing part of the equipment and, therefore, by the extent of solubilization of the SC-CO2 in the liquid solution.
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In summary many different techniques were developed based on the characteristics of the material to be treated and/or the final product. This underscores the versatility of the SCF-based techniques: a process limitation in one area often opens up new research and solutions in other directions. For instance, if the starting material is soluble in the SCF the RESS will be preferred while in case of the low solubility an antisolvent process will be used. When organic solvent has to be avoided, i.e. in the case of biological products, processes such as CAN-BD or SEDS with three co-axial nozzle should be selected. Finally, the characteristics of the desired product (micronized, coated, encapsulated, etc.) would drive the selection of the technology as well.
APPLICATION OF SUPERCRITICAL FLUID IN PHARMACY FIELD:
1. DRUG DELIVERY A) Particle and Crystal Engineering (Size Reduction and Solid State Chemistry) B) Particle Coating C) Particulate Dosage Form Cyclodextrin Inclusion Complexes Extrusion Liposomes Preparation Microspheres 2. Sterilization 3. Solvent Removal 4. Extraction 5. Supercritical and Subcritical Chromatography
DRUG DELIVERY: A) Particle and Crystal Engineering (Size Reduction and Solid State Chemistry) Standard Micronisation Processes: Comparison with Supercritical Fluid base Technique The standard micronisation processes comprise crushing/ milling, air micronisation, sublimation and recrystallization from solvents. These techniques can often undergo several practical problems. The mechanical treatments can damage, degrade Page 12

particles due to high stresses (thermal and mechanical) generated by attrition. Moreover, changes of drug crystallinity could originate during stress, leading to chemical or physical instability or to an increase of surface energy with increased tendency to adhesion and agglomeration. In the conventional crystallisation process the products are more or less contaminated with the solvents and waste streams are produced. An alternative particle engineering technique is the spray drying process that can partially alleviate some of the problems of the mechanical techniques, but it is difficult to use with poorly water-soluble drugs and usually requires high operating temperatures, which could cause degradation of heat sensitive materials. Furthermore, all these techniques share the disadvantage of a poor control of the particle size distribution. Some of the practical problems associated with the use of conventional micronisation processes can be overcome by means of supercritical fluids technologies, allowing the preparation of micro- or even nanoparticles with a narrow particle size distribution.
The main established advantages of supercritical CO2 based techniques are: Mild operating temperatures; Single-step process; Recovery and recycle of fluid; Green technology; Solvent-free products. Furthermore, traditional methods for the production of drug microparticles require numerous manufacturing steps that can be avoided, in case of SCF techniques, bringing to a simplification and better control of the process (Fig. 8).
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FIG.-9 Process simplification by SCF relative to conventional micron-sized crystalline powder production.
Crystallization is a transient non-equilibrium process associated with a phase change leading to crystal formation. The driving force for this process is supersaturation. A high degree of supersaturation is generated in non-equilibrium conditions, in the area above the saturation boundary in the concentration/temperature diagram (spinodal curve, Fig. 8). In this area, the crystallization process, referring to the simultaneous occurrence of nucleation, crystal growth, and agglomeration, takes place. Traditional crystallization methods include sublimation, crystallization from solutions, evaporation, thermal treatment, desolvation, or grinding/milling. The crystallization process is governed by both thermodynamic and kinetic factors, which can make it highly variable and difficult to control. For a compound existing in various solidstate forms, thermodynamic factors influence the conditions and direction in which a transformation from one polymorph to another takes place, while kinetics influences the time in which this transition occurs. The relative thermodynamic stability of solids and the driving force for a transformation at constant pressure is given by the Gibbs equation:
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FIG.-8 Spinodal curve: A) undersaturated area, decreasing temperature, the solution

goes to the saturation B), C) solution enters the metastable zone, D) fast nucleation, E) concentration decreases with crystal growth.
The relative stability is given by the algebraic sign of G; if it is negative the phase transformation occurs naturally and the change continues as long as the free energy of the system decreases. G=0 when the system is in equilibrium with respect to transformation; G is positive when the free energy increases and the transformation does not occur. The operative conditions that can be changed to control the process are temperature range, cooling/heating rates, choice of solvents, and variation of solute concentration (which depends on the temperature). Additional factors such as impurity level, mixing regime, vessel design, and cooling profiles can deeply impact the size, number, and shape of the crystals produced. With respect to traditional processes, the use of SCFs implies the possibility of more precise control of the operative conditions, but also of taking advantage of some peculiar conditions, such as pressure, rate of solvent evaporation and fine-tuning of the density. As discussed in some detail below, the kinetics of the crystallization process is mainly driven by diffusion. Therefore, the density of the solvent greatly impacts this aspect of the phenomenon, as this parameter strongly influences the diffusivity of the solvent, or the antisolvent and, therefore, on the transient concentration gradient. Additionally, the possibility of modulating the rate of solvent evaporation deeply influences the kinetics of the process because of its relation to the rate of accumulating supersaturation. Page 15

RESS is an attractive process, simple and relatively easy to implement on a small scale when a single nozzle is used. The main limitation of the use of RESS technique is the poor solubility of pharmaceutical products in supercritical CO2. The low solubility can, in some instances, be overcome with appropriate tuning of the fluid density and the use of an adequate cosolvent. Other limitations are poor predictive control of particle size, morphology along with the difficulty to scaling-up the process because of particle aggregation and nozzle blockage caused by cooling effects due to the rapid expansion of the supercritical solution. Analogous RESS experiments were performed with supercritical CO2 and ibuprofen to investigate the effects of extraction pressure, pre-expansion temperature, capillary length, spraying distance, and collision angle on the size and morphology of the precipitated particles. Micronisation of ibuprofen was successfully obtained with average particle size of the powder below 8m all the different conditions tested. No clear dependence of particle size on the pressure (1317MPa) was found, while a slight decrease in the particle size was observed increasing the pre-expansion temperature. The particle size decreased also when the capillary length (812mm) and the collision angle (from 45 to 90) were increased, and the spraying distance (26 cm) was decreased. RESS is usually performed on a binary system made up by the supercritical fluid and the solid. Sometimes a cosolvent can be added to improve the solvent power of the SF. Polar organic solvents or capable of H-bonding (e.g. methanol) can enhance the solubility of polar solutes in supercritical CO2. Enhancement factors (solubility in the SFcosolvent system divided by the solubility in the SF alone) values of 104106 are common and even values as high as 1012 have been reported. Besides cosolvents, also cosolutes can enhance the solubility of the solute in supercritical CO2. The effects of addition of benzoic acid (BA) on the solubility in supercritical CO2 of salicylic acid or phenanthrene were analyzed. The solubility of both compounds was enhanced by the presence of BA while the crystal size decreased.
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TABLE-2 Volume diameter at 50% frequency (D[0.5]) and span for ASA powders obtained by RESS at different temperature and pressure values (S.D.), n=3 Experimental Conditions: T(.C), P(MPa) 40-15 40-20 40-25 50-15 50-20 50-25 5.39(0.06) 4.75(0.06) 4.02(0.53) 4.70(0.16) 4.60(0.40) 4.04(0.93) Untreated Acetylsalicylic Acid D(0.5)=520m 1.89(0.27) 1.79(0.05) 2.94(2.05) 1.37(0.19) 3.40(1.25) 1.28(0.76) D(0.5) (m) Span
Micronized particles of acetylsalicylic acid (ASA) were obtained with the RESS process. The solid state characteristics of micronized ASA, as well as the correlation between process parameters, namely temperature and pressure, and solid-state properties were investigated. A remarkable reduction of particle size was obtained, which linearly correlated to pressure (Table-2), while temperature apparently did not influence the particle size of the products. No polymorphism was induced by the RESS process, although a significant melting temperature decrease for micronized ASA was found and linearly correlated with the reciprocal of the mean particle radius, by applying the GibbsDuhem-Laplace equation. GAS/SAS Process Different SAS injection rates were used with sub- and supercritical carbon dioxide to recrystallize sulfamethizole from solutions in acetone or N, Ndimethylformamide (DMF). Sulfamethizole crystals with tabular and platy habits were produced by different operating conditions; in particular, low CO2 injection rates produced large crystals with irregular tabular habit, while higher injection rates gave rise to small particles with thin platy habit. Also the solvent choice affected the crystal habit: large platy crystals, independently of injection rate, were obtained from DMF; low injection rates afforded tabular crystals, whereas higher injection rates resulted in thin
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platy crystals from acetone. Ultrasounds application to the nozzle was investigated: 22.5 kHz sonication accelerated nucleation, producing a larger number of small crystals. Particle size increased with temperature increase, both in sub- and supercritical conditions. The effect of pressure on particle size depended on the solvent choice: from ethanol and ethanol/methanol solutions, a significant decrease of particle size with pressure increase was observed, while the opposite occurred from acetone solutions. The different behavior was explained taking into account the difference in vapor pressure of the solvents and the different phase equilibria of the ternary system. In case of lower solution flow rates (0.3 L/h), more concentrated solutions afforded smaller particles, whereas with high flow rates (0.6 L/h) the reverse tendency was found. The particle size was more or less independent of the nozzle diameter when similar nozzles were used. The nozzle design affected the particle size: for example, using a two-flow nozzle, instead of a Laval nozzle, the mean particle size was reduced, probably because the twoflow nozzle enhanced atomization energy and mixing efficiency. ASES Process Insulin, albumin, lysozyme, and recombinant human deoxyribonuclease (rhDNase) were precipitated using the ASES process and CO2 modified with ethanol. The proteins precipitated as nano-sized particles ranging from 100 to 500 nm, and their physical properties and biochemical stability depended on the operating conditions. SEDS Process The influence of SEDS process parameters mentioned above on the recrystallization of the model drugs budesonide and flunisolide. As for budesonide, the starting material was composed of irregularly spherical particle aggregates (typical morphology of micronized powders with high energy operations). SEDS crystals formed at 10MPa and 80 C from acetone were smooth, discrete and nearly spherical, while crystals from methanol had a plate-like shape. The diameter of the particles from acetone was within the 13m range, while those from methanol had a diameter within 530m. The higher solubility in the SF of acetone compared with methanol influences the saturation profile with a consequent effect both on nucleation and crystal growth, producing particles with different size distributions and morphologies. The differences Page 18

found for samples obtained from acetone or methanol could be partly due also to bond formation between solvent and drug, thus affecting the extraction process. As demonstrated by their uniformity, particle size and morphology can be effectively controlled by the SEDS process. These examples confirm that changes in morphology, size, and surface properties of powders can be manipulated and designed by supercritical fluids processes. In addition to morphology and micromeritics above discussed, particle engineering of pharmaceuticals must take into account also another very important topic, with both technological and biopharmaceutical implications: polymorphism,
pseudopolymorphism and solidsolid phase transitions. The kinetics of the phase transitions can be influenced by typical environmental parameters (temperature, pressure, relative humidity), presence of crystalline defects, impurities and mechanical stress. Polymorphic purity is an important parameter to consider in drug products, since the presence of differing crystal phases can accelerate the conversion process by lowering the relevant activation energy barrier. The phase transformation could lead to different polymorphs with unwanted physical and chemical properties; for this reason, regulatory authorities have long recognized the need for limiting polymorphic impurities in pharmaceutical materials. Supercritical carbon dioxide in control of polymorphism As explained in the previous section, different polymorphs have different physical characteristics; thus, the identification of stable polymorphs with desired physical properties is very important for drug products development. The solid phase must be monitored, especially after technological unit processes potentially able to modify the solid-state properties of the components. In particular, during the conventional micronisation process such as milling, spray-drying and freeze drying, the substances are usually exposed to mechanical stress, contact with solvents, heating cooling cycles, that can often lead to alterations of the solid phase, such as new polymorph formation, dehydration, vitrification via solid-state or melting mechanism. The treatment with supercritical CO2 of a new alkylating anticancer drug, at 55 C and 40MPa, originated a new crystal phase via a solution-mediated process.
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Carbamazepine polymorphs were also investigated in order to obtain the pure crystalline phase stable at ambient temperature (form III) by means of supercritical CO2. An equimolar mixture of carbamazepine polymorphs I and III was processed with supercritical CO2 to obtain a crystallographically pure phase. It has been proved that the suspension in supercritical CO2 leads to an almost quantitative conversion of form I into form III (Table 3). The enrichment of the mixture in terms of form III is essentially due to the conversion of form I via solubilization in supercritical CO2 followed by recrystallization of the less soluble and more stable polymorph. In another example, deoxycholic acid powders were stored in a pressure vessel purged with carbon dioxide at 12MPa and 60 C for fixed time intervals. After 1h, new peaks in the powder X-ray diffraction pattern suggested that a new polymorph was being generated. The conversion was almost quantitative after 6h treatment. Table 2 Percent of carbamazepine form III obtained after static treatment of a 1:1 mixture form I: form III in supercritical CO2 at 35MPa and 55 C (n=4) SF Treatment (h) 0 6 9 23 48 %Form 3(S.D.) 47.9(3.9) 88.3(0.9) 90.6(2.8) 91.2(4.7) 94.7(0.7)
SEDS Process Salmeterol xinafoate (SX) is an anti-asthma drug administered by inhalation and thus requiring particle size within 25m. Commercial SX, generated by a conventional crystallization process, has very poor flow properties, and it is unsuitable for size reduction with a fluid energy mill. Two different approaches to SX recrystallization as fine particles, i.e. fast cooling crystallization and the SEDS process, were investigated. In the fast cooling crystallization, SX was first dissolved in a hot organic solvent and then quenched in a chilled solvent to induce supersaturation and consequent precipitation of the drug. A wide range of solvents and experimental Page 20

conditions was examined: SX with good flow properties, suitable for further milling down to 25m-sized particles, was obtained. In the second approach, solutions of SX in ethanol and in acetone were introduced in the SEDS apparatus in differing working conditions (i.e., temperature, pressure, solution concentration, and supercritical carbon dioxide flow rates). This process was able to provide, without extra milling operation, microfine free-flowing crystalline particles (25 m), virtually free from retained solvent. Interestingly, the SEDS process, unlike conventional crystallization
procedures, was able to produce specific polymorphs of SX, depending on the operating conditions. The fast cooling crystallization gave exclusively SX form I, namely the stable polymorph at ambient temperature. Moreover, micronised SX obtained by SEDS showed superior crystalline purity, with respect to commercial micronized batches (MSX, form I). Surface thermodynamic properties of both solid phases (obtained by SEDS or MSX) were compared by inverse gas chromatography. Metastable SX form II (prepared by the SEDS process) exhibited higher surface entropy and free energy and a more polar surface than the stable form I (also prepared by the SEDS process), whereas MSX displayed a higher surface free energy and enthalpy than SX form I. B) PARTICLE COATING Conventional coating processes are carried out by nebulizing an aqueous or organic solution of the coating material (sugar, polymer, wax) onto the solid dosage form. The use of aqueous solutions eliminates many disadvantages associates with organic solvents; however, it increases the drying time due to the higher latent heat of vaporization of the water relative to organic solvents. Furthermore, the number of the material, in particular polymers, that can be dispersed or dissolved in water is limited. Last but not least, the coating of the small particles still represents an issue. Solvent less coating technologies may overcome some of the above-mentioned disadvantages, and reduce the overall cost by eliminating the slow and expensive process of solvent removal. A fluidized-bed coating process based on the RESS was described by Tsutsumi et al. The coating granulation process consists of three main steps: extraction, expansion and fluidization. In a typical experiment, CO2 flows through the melted coating material in an extraction column, where the CO2 becomes saturated with the solute. The bed is fluidized by adding pure CO2 via a bypass line; thus the CO2 acts as Page 21

carrier fluid as well. The CO2, saturated with the coating material, expands through a nozzle into the fluidized bed, which consists in a column installed inside an autoclave. The solid final product is separated from the gas by a cyclone and several filters. The column is equipped with two pressure sensors: one at the bottom and one on the top of the column. These allow the control of the pressure drop across the fluidized bed and calculation of the minimum fluidization velocity. Below this last the bed is packed, while above the bed expands and the particles move. Process key parameters are temperature, pressure, solidification kinetics of the coating material, and fluidization gas velocity. The relatively low pressure and temperature as well as the absence of organic solvents enable the use of such process for encapsulating sensitive material like proteins. These authors tried to coat with paraffin irregularly shaped particles of bovine serum albumin (BSA) and insulin, by means of RESS process in a high-pressure fluidized bed running with SC-CO2. The fluidization behavior of BSA was improved by adding free flowing lactose. Lactose was mixed with BSA in different volume percentages and the minimum fluidization velocity of each mixture was measured at fixed pressure and temperature (812MPa and 4055 C) by stepwise increasing the SC-CO2 flow rate. It was noticed that the minimum fluidization velocity decreased by increasing pressure as well as by decreasing temperature; similar effects were found also by. The obtained coated particles were tested for dissolution behavior in comparison to the uncoated particles. Dissolution retardation of even more than 180min, indicating improved encapsulation, was achieved for certain experiments. Although this technique appears to be very promising, to date only few experiments have been done and too few data are available to express an opinion about its future application in pharmaceutics. A supercritical fluid coating process by using a simple autoclave equipped with an rotating impeller was used to prepare BSA microparticles coated with two lipids. Protein particles were coated with trimyristin (Dynasan 114) and Gelucire 50-02, two glyceride mixtures with a melting point of 45 and 50 C, respectively. Dynasan 114 afforded a discontinuous coating constituted by disordered micro-needles. This led to a drug release kinetics exhibiting a significantly high initial burst (35% in 5 min); then 70 and 85% of the protein was released in 30 min and 5 h, respectively. On the other hand, a Page 22

prolonged protein release (over 24 h) was achieved with particles coated with Gelucire 50-02, which produced a more homogeneous film coating. It was also shown that BSA did not undergo any degradation following the SC-CO2 treatment. Noteworthy, lipids are quite soluble in pressurized CO2, while the most commonly used coating materials including many polymers show very low solubility in this supercritical fluid. This problem can be overcome by adding a co-solvent to the coating solution. Compared to conventional techniques the amount of organic solvent used here is greatly reduced. Patented an anti solvent process to prepare microcapsules containing active ingredients with a polar polymer film (polysaccharide, cellulose derivates, acrylic or methacrylic polymers, polymers of vinyl esters, polyesters, polyamides, polyanhydrides, polyorthoesters, or polyphosphazenes). The procedure implied the suspension of the active ingredient in an ethanol solution of the polymer, followed by mixing with SC-CO2 for the ethanol extraction. Mishima et al. used a method called rapid expansion from supercritical solution with a non-solvent (RESS-N) for the microencapsulation of proteins (lipase and lysozyme) with polymers such as polyethylene glycols (PEGs), poly methyl methacrylate (PMMA), polylactic acid (PLA), polylactide-co-glycolide (PGLA) and PEG PPG(polypropylenglycol)PEG triblock copolymer. The polymer, protein and ethanol were placed inside an autoclave, then the mixture was stirred, and the CO2 pumped in at fixed temperature and pressure. Non-agglomerated proteins containing microparticles were obtained upon the expansion of the polymeric solution. The thickness of the polymer coating, the mean particle diameter as well as the particle size distribution, could be controlled by changing the polymer feed composition.
C) PARTICULATE DOSAGE FORM: 1) CYCLODEXTRIN INCLUSION COMPLEXES: Cyclodextrins are cyclic oligosaccharides able to fully or partially include in their hydrophobic internal cavity a guest molecule of appropriate size. This complexation allows to improve some physico-chemical properties such as solubility, dissolution rate,
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Stability, as well as organoleptic characteristics. Conventional preparation methods of CDs inclusion complexes include kneading, co-precipitation, co-evaporation, cogrinding, and freeze- and spray drying. Studies on the feasibility of supercritical fluid processes to produce inclusion complexes between drugs and CDs in the solid-state are reported in literature. A successful complexation (94% inclusion yield) between piroxicam and -cyclodextrin (-CD) was obtained by. The inclusion experiments were performed by keeping a physical mixture of -CD and piroxicam for 6 h in contact with CO2 at 150 C and 15MPa without the use of organic solvents. By using a similar method Moneghini et al. tried to include nimesulide into -CD. The obtained results indicated that the drug was only partially included. However, the physico-chemical characterization of the obtained product pointed out the existence of interactions between drug and carrier that led to an increased in vitro drug dissolution rate. Charaoenchaitrakool et al. prepared a complex of methyl--cyclodextrin (M--CD) and ibuprofen affording the M--CD solid/liquid transition as a consequence of the interaction with SC-CO2. The same research group used an ASES process for producing micron-sized naproxen formulation incorporating hydroxyl propylated- and methylated-CD. A controlled particle deposition process using SC-CO2 (CPD) was developed by Turk et al. to produce complexes of racemic ibuprofen and -CD. In this method, ibuprofen and -CD were filled in to two separates cartridges as pure compound and in another cartridge, as a physical mixture: the cartridges were inserted in a high-pressure cell heated and pressurized at the desired values (40 C and 25 or 30MPa). After 15 h under stirring, the system was depressurized within 10 min. The maximum inclusion yield, in the case of separate cartridges was 88%, and 60.5% in the case of the physical mixture. Both the dissolution rate coefficient and the dissolved amount after 75min of the CPD complexes were found to be significantly higher than those of unprocessed ibuprofen alone and ibuprofen/-CD physical mixture. A SEDS process (10MPa, 4080 C) was carried out to produce crystalline channel-type and amorphous -CD particles and crystalline channel-type -CD complexes in a singlePage 24

step process. The increase of process temperature changed the crystallinity of -CD. In particular, at 80 C amorphous -CD was obtained, while the complexes crystallized a tetragonal channel-type form to hexagonal channel-type form. The dissolution behavior of budesonide/-CD complexes depended on their crystal structure: the tetragonal dissolved faster than hexagonal form. This represents a nice example of -CD crystallinity change with subsequent variation of the dissolution rate of complexed budesonide stemming from the modification of the operative process conditions. SAS process was used for the preparation of an inclusion complex between simvastatin (SV) and hydroxypropyl--cyclodextrin (HP--CD). The activity of the SV/HP--CD Complexes was tested in vivo in comparison with uncomplexed simvastatin. SV/HP-CD inclusion complex performed better than SV in reducing total cholesterol and triglyceride levels; this could be primarily attributed to the improved solubility and dissolution of the complex. In another study, the feasibility of the ASES process for preparing solid-state inclusion complexes of itraconazole (ITR) with HP--CD was investigated. ITR/HP--CD complexes with a significantly higher dissolution rate (90% of ITR dissolved within 510min) than the unprocessed ITR and its physical mixture were obtained. 2) EXTRUSION: The above-mentioned capability of SC-CO2 to plasticize polymers at low temperature can be exploited in the extrusion process. Here, the SC-CO2 can both change the rheological properties of the material, and behave as an expansion agent. The dissolution of a large amount of SC-CO2 determines a polymer expansion and viscosity reduction. The viscosity reduction results in lower mechanical constraints and decreases the required operating temperature, thus allowing processing of thermolabile compounds. Verreck and co-workers carried out extrusion of PVP-VA (polyvinylpyrrolidone-co-vinyl acetate), Eudragit and ethylcellulose, by means of a twin-screw extruder in which pressurized CO2 was injected at a constant pressure rate. The physico-chemical characteristics of the polymer before and after injection of CO2 were evaluated. The specific surface area and the porosity of the polymers increased after treatment with carbon dioxide, eventually resulting in enhanced polymer dissolution in water. Page 25

The expansion of the CO2 at the nozzle determined a change of the morphology of the system toward a foam-like structure. The same observations were done in the case of itraconazole/polymer combinations as well. Another work illustrates the use of SC-CO2-assistedextrusion for the preparation of a hot melt extruded monolithic polymer matrix for oral drug delivery. The polymer used was polyethylene oxide (PEO) while the model drug was carvedilol. The SC-CO2-assisted process gave rise to extruded material with faster dissolution compared to that obtained with a classical extrusion process. In conclusion this represents an interesting example of process
contamination where the introduction of a SCF technology into a conventional technique leads to a better performing material. 3) LIPOSOMES PREPARATION: The preparation of stable liposomes formulation on industrial scale is still amajor issue in pharmaceutics mainly due to the need of the large amount of organic solvents and the high energy consume. In this respect, SCF-based technologies have attracted a great deal of interest during the past 10 years, as a green alternative to classical methods. Pioneering work in this field was done by Frederiksen and coworkers, based on amodification of RESS process. The apparatus was mainly composed by two parts: a high-pressure system in which phospholipids and cholesterol were dissolved into the supercritical phase and a low pressure system in which the supercritical phase (containing also a various amount of ethanol) was expanded and simultaneously mixed with a water phase containing dextran and fluorescein isothiocyanate to yield liposomes. Liposomes with a diameter of 200 nm were obtained. Noteworthy, this technique get the same encapsulation efficiency but required 15-fold less organic solvent, compared to the ethanol injection method of Batzri and Korn (1973). Castor and Chu (Castor, 2005; Castor and Chu, 1998) presented a Supercritical FluidsTM CFN (SFS-CFN) apparatus formaking liposomes containing hydrophobic or hydrophilic drugs featuring critical, subcritical or near-critical CO2. Such fluids were used to solvate phospholipids, cholesterol and other raw materials. After a specific mixing time the resulting mixture was decompressed by means of an injection nozzle into a chamber that contained phosphate-buffered saline or another Page 26

biocompatible solution with the hydrophilic drugs (recombinant proteins, RNA, DNA). SCF depressurization and phase-conversion into a gas (with formation of bubbles) created a deposition of the phospholipids in the aqueous phase and, consequently, the formation of phospholipid bilayers encapsulating the hydrophilic drug. In the case of hydrophobic drugs, the phospholipids and the target compounds were solvated simultaneously in a SCF cocktail which was dispersed continuously into the aqueous environment. In a third variation of this technique, hydrophobic drugs and phospholipids were directly solvated in the SCF prior to injection into an aqueous solution. The characteristics of the obtained liposomes depended on several process parameters: size and design of the nozzle (affecting the bubble sizes and therefore the size of liposomes formed); decompression rate which defines the characteristics of the deposited phospholipids as well as the intensity of the mixing (slow decompression produced smaller and more uniform liposomes); interfacial forces between the SCF and the aqueous phase; charge distribution of the liposomes and its interaction with the surrounding aqueous medium (pH, ionic strength and electrolyte composition); the nature of compounds being encapsulated. It was found that liposomes formed with a 0.5mm nozzle with lecithin at28MPa and60 Chad an average diameter of 478nm with unimodal distribution, while liposomes formed with 0.6mm nozzle under identical condition had an average diameter of 326 nm. The incorporation efficiency varied between 1% for small unilamellar vesicles and 88% for some multilamellar vesicles (Castor and Chu, 1998). The stability of liposomes was also tested, by measuring the particle size distribution as a function of time. SC-CO2 liposomes proved to be stable at 4 C over a 6-month period. This technique also afforded liposomes incorporating molecules such as paclitaxel, camptothecin, betulinic acid, bryostatin 1, vincristine and doxorubicin, with diameter between 100 and 300 nm. In vitro and in vivo results suggested that these formulations are more effective than the commercial available ones. More recently, other authors (Otake et al., 2001) produced liposomes by using the method described by Castor and Chu. Phospholipids and ethanol were introduced in a view cell, along with the CO 2. The cell temperature was then raised up to 60 C, namely a temperature higher than the phospholipid phase transition temperature, while the pressure was kept at 20MPa. An aqueous dispersion of liposomes Page 27

was obtained through the formation of an emulsion by introducing an aqueous glucose solution into the cell. Then, the pressure was reduced to release the CO2, leading to a homogeneous liposomial dispersion. Transmission electron microscopy indicated that the vesicles obtained were large ellipsoidal unilamellar liposomes with a diameter from 0.1 to 1.2m, enabling a high entrapping efficiency (up to 20% for hydrophilic substances and 63% for hydrophobic compounds). The same method was used to prepare chitosancoated cationic liposomes for DNA transfection (Otake et al., 2006). Pressurized CO2 forms carbonic acid in contact with water thus it lowers the water poly methyl
methacrylate, swollen with CO2 (Sproule et al., 2004) The experiments were carried out with a view cell that allow in situ quantification of the polymer swelling by laser dilatometry. Foaming was studied by means of a X-ray computed tomography, while protein impregnation was verified by confocal microscopy. Finally, Barry et al. produced bovine chondrocytes containing methacrylate scaffolds. Polymer foaming was achieved by placing PEMA/THFMA discs under CO2 at 40 C and 10MPa for a time ranging from 1 to 48 h, then by rapidly depressurizing the system (Barry et al., 2004). The study demonstrated that the change in the structure of the substrate from flat disk to foam enhanced the cell phenotype retention, although the authors concluded that further modifications in the SC processing were required to obtain optimum porosity for cell migration and cartilage tissue formation.
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FIG.-9 Process Flow Sheet of Liposome
The 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol were dissolved in supercritical carbon dioxide modified with ethanol. Rapid expansion of the supercritical solution into an aqueous phase containing a marker results in the formation of liposomes encapsulating the marker (Fig. 9). Liposomes have an average size of approximately 4050 nm. 4) MICROSPHERES: Microparticles have a variety of structures (Fig. 10): Particles with irregular geometry, composed of an active substance in form of aggregates or molecularly dispersed solid embedded in to a matrix. They are called microspheres; Particles with spherical geometry, composed of a core of active substance surrounded by a solid polymeric or proteic shell. They are called microcapsules. There is no universally accepted size classification of these particles.
Page 29
FIG.-10 Structure of the Different Microparticles.
However, many workers classify spheres/capsules smaller than 1m as nanospheres/capsules and those larger than 1000m as macro-spheres/capsules. Commercial micro-spheres/capsules typically have a diameter between 3 and 800m and contain 10 90 wt.% carrier/core. A wide range of materials has been embedded/encapsulated, including adhesives, agrochemicals, live cells, active enzymes, flavors, fragrances, pharmaceuticals, and inks. Most carrier/shell materials are natural or synthetic organic polymers, but fats and waxes are also used. Examples Debenedetti et al. obtained microparticles by the RESS co-precipitation of a drug (lovastatin) and a biodegradable polymer (poly(D, l-lactic acid) (DL-PLA)).The co-precipitation of the polymer and the drug led to a heterogeneous population of microparticles consisting of microspheres containing a single lovastatin needle, larger spheres containing several needles, microspheres without protruding needles and needles without any polymer coating. Mishima described the formation of microspheres of flavones and a polymer (Eudragit- 100 or PEG 6000) by spraying at atmospheric pressure, a suspension of flavonoids in a supercritical solution of the polymer and a co-solvent. Sze Tu et al. used the ASES technique for the coprecipitation of a model drug, parahydroxybenzoic acid (p-HBA) with the biodegradable polymers, poly(lactideco-glycolide) (PLGA) and poly(l-lactic acid) (PLA) as presented on Fig. 12. A multiple nozzle assembly arranged coaxially was designed for the co-introduction Page 30

of the drug and polymer solutions (Fig. 11). In this configuration, solutions are expanded at a much faster rate than in the conventional ASES process since minute volumes of the solution continually dispersed by the nozzle suddenly enter a region of high anti-solvent concentration. The rapid expansion causes the solid to precipitate out of solution almost instantaneously. In this case, supercritical fluid is used only for its anti-solvent properties. Coprecipitation of the p-HBA and PLGA resulted in the formation of p-HBA particles coated with the polymer microspheres. The coprecipitation of p-HBA with PLA resulted in the formation of a product where the drug and polymer were incorporated together in a fibrous network.
FIG.-11 Process flowsheet by Sze Tu et al.
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References:
1) Bansal A. K., Kakumanu V. K., Supercritical fluid technology in pharmaceutical research. Business Briefing: Labtech 2004. 70-72. 2) Pasquali I., Ruggero B., Are pharmaceutics really going supercritical?, International Journal of Pharmaceutics. 364(2008), 176-187. 3) Giordino F., Pasquali I., Ruggero B., Solid-state chemistry and particle engineering with supercritical fluids in pharmaceutics. European Journal of pharmaceutical sciences, 27(2006), 299-310. 4) Fages J., Lochard H., Letourneau J. J., Sauceau M., Rodier E., Particle generation for pharmaceutical applications using supercritical fluid technology. Powder Technology, 141(2004), 219-226. 5) Giordino F., Pasquali I., Ruggero B., Supercritical fluid technologies: An innovative approach for manipulating the solid state of pharmaceuticls. Advanced Drug Delivery Reviews, 60(2008), 399-410. 6) York P., Strategies for particle design using supercritical fluid technologies. Pharmaceutical Science and Technology Today, Volume-2 Number-11, 1999, 430440. 7) Kiran E., Yeo S. D., Formation of polymer particles with supercritical fluids: A Review. The Journal of Supercritical Fluids, 34(2005), 287-308. 8) Perrut M., Jung J., Particle design using supercritical fluids: Literature and patent survey. The Journal of Supercritical Fluids, 20(2001), 179-219. 9) Giordino F., Bonassi L., Ruggero B., Castoro V., Rossi A., Zema L., Gazzaniga A., Solubility and conversion of carbamazepine polymorphs in supercritical carbon dioxide. European Journal of pharmaceutical sciences, 13(2001), 281-286.
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Supercritical Fluid and Subcritical Fluid

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SUPERCRITICAL FLUID AND SUBCRITICAL FLUID