Supercritical fluids (SFs) are gases and liquids at temperature and pressure above their critical points (Tc, critical temperature; Pc, critical pressure). SFs exist as a single phase with several advantageous properties of both liquids and gases. They have density values that enable appreciable solvation power and the viscosity of solute in SF is lower than in liquids. Furthermore, solutes have higher diffusivity, which allows high mass transfer [14]. CO2 is the most widely used SF for pharma- ceutical applications because of its low critical temperature (31.2°C) and pressure (7.4 MPa); it is non-flammable, non-toxic and inexpensive. Depending on the nature of the process, SFCO2 can serve either as a solvent or anti-solvent. To increase the salvation power of an SFCO2 an organic modifier such as acetone or ethanol could be added to the fluid.
SF can be applied to protein particle design (Fig. 7.2) in three broad ways
● Precipitation from supercritical solution composed of SFCO2 and protein
● Precipitation from gas saturated solutions
● Precipitation from saturated solutions using SF as anti-solvent
In the first method, the drug is dissolved in the SF and followed by rapid expansion of the SF solution across a heated orifice to cause a reduction in the density of the solution and reducing the salvation power of the SF, which leads to the precipita- tion of the drug [16]. This process is termed the ‘rapid expansion of supercritical solution’ (RESS).
The RESS process depends on sufficient solubility of the material to be processed in the SF while the solubility of the material depends on the density of the SF, the drug’s chemical structure and the SF-drug contact time. The mor- phology and the size distribution of the particles formed could be influenced by the pre-expansion concentration of the solute in SF and the expansion conditions (e.g. temperature and pressure). The higher the pre-expansion concentration, the narrower the particle size distribution. The expansion conditions depend on the temperature, the geometry and size of the nozzle.
The limitations facing the use of RESS for proteins include high temperature needed for the rapid expansion, which could destroy proteins, poor predictive control of particle size and morphology, scale up limited by particle aggregation and nozzle blockage caused by expansion cooling. The solubility of the drug in SFCO2 also constitutes a major limitation.
Vent
Mag DriveBack pressure regulator Particle Collection
Pre-expansionSpray Device Pre-expansion
BPRVent Vent
Back Pressure Regulator Reaction Vessel Particle formationParticle formationParticle formation
SCF Molten solids Particle formation
Organic solutionOrganic solution
Organic solution
Coaxial nozzle
Horn Organic solution Vent
BPR BPRVentVent
Vent BPR
BPR
SCF
SCF SCF
SCF SCFSCF SCF SCF SCF SCF SCF
SCF
Static Supercritical Fluid Process Batch SAS/ASES/PCAContinuous SAS/ASES/PCASEDSSAS-EM
RESSPGSSGAS
Expanded Organic solution Particle formationSpray Device Particle Collection
Extractor Fig. 7.2Schematic of various applications of SF technology in particle design, reprinted from ref. 15 with permission from Elsevier
The second method is quite harsh but similar to RESS process as they both involve use of SFCO2 as a solvent rather than an anti-solvent. This process involves dissolving the SF in molten solute and the resulting supercritical solution fed via an orifice into a chamber to allow rapid expansion under ambient conditions [17]. The dissolved gas decreases the viscosity of the molten compound and so the gas saturated liquid phase is expanded to generate particles from materials that are not necessarily soluble in SF. The presence of the CO2 allows the material to melt at temperature significantly lower than the normal melting or glass transition temperature.
Precipitation from saturated solutions using SF as antisolvent, takes advantage of the limited solvation power of SFCO2 for proteins. This method utilizes a similar concept to the use of anti-solvent in solvent based crystallization processes. The high solubility of SFCO2 in organic solvents leads to volume expansion when the fluids make contact. This leads to reduction in solvent density and subsequent fall in salvation capacity. This leads to super-saturation, solute nucleation and particle formation.
Different variants of the use of SF as anti-solvent have been developed and all have been successful at producing aerodynamic and stable protein particles. These include:
● Gaseous antisolvent (GAS)
● Aerosol solvent extraction system (ASES)
● Solution enhanced dispersion by SF (SEDS)
● Precipitation by compressed antisolvent (PCA)
5.1 Gaseous Antisolvent
This process involves adding SF to a particle formation vessel containing the protein solution of interest. The protein precipitates during the dissolution of SF in the solvent. Generally, the SF is introduced through the bottom of the vessel and bubbled through the protein solution to achieve better mixing of the solvent and anti-solvent. Once the protein has precipitated out, the solvent-SF can be removed. The protein particles can then be washed with a sequence of SF washes.
Following the wash, the pressure in the vessel is released and the protein powder removed.
It is important that the solvent in which the protein is dissolved has a high solvent power for the protein, preferably be soluble in the SF of choice and be compatible with the protein. Organic solvents such as dimethyl sulphoxide and dimethyl formaldehyde have been used because they meet the criteria of salvation of the protein and solubility in SFCO2. However, toxicity and compatibility of these solvents with protein molecules remain an issue [18].
Although SFCO2 remains the promising SF for pharmaceutical use, alternatives such as ammonia and ethane have been studied. While ammonia produced completely
denatured proteins, ethane produced results comparable to SFCO2 in terms of particle size and improved biological activity of insulin. The limitation facing the use of SF-ethane is its high flammability.
Studies have shown that the type of solvent used has effect on the particle size, morphology and biological activity of the molecule [19, 20]. The principal disad- vantage of this process is the lack of control over particle formation. This has been observed to be true in batch operating conditions because the level of saturation is not maintained.
5.2 Aerosol Solvent Extraction System
Unlike in GAS method where the SF is pumped into the protein solution, the opposite happens in the ASES process. The CO2 is pumped into a high pressure vessel until the system reaches the desired fixed conditions (pressure and temperature). The protein solution is then sprayed via an atomization device into the vessel containing SFCO2. Precipitated particles are then collected on a filter at the bottom of the vessel. This process retains the antisolvent concept of GAS but this happens at the droplet level. This offers a favourable higher antisolvent to solvent ratio, an increased surface area and mass transfer rate, and hence, an acceleration of the drying process. The rate is particularly fast when the operating pressure reaches the mixture critical pressure. The process is then controlled by mixing of miscible fluids rather than mass transfer over the interface of the droplets in the spray [21].
There is a need for special attention for mass transfer when the miscibility of the fluids is poor especially in systems containing water and CO2. The mass transfer can be improved by increasing the drying medium to solvent ratio, decreasing droplet size or relative velocity between the solvent and drying medium. ASES enables production of protein particles with narrow size distribution, uniform shape and desired physico-chemical characteristics.
5.3 Solution Enhanced Dispersion by SF
To minimise particle agglomeration often observed in ASES and other antisolvent based techniques and to reduce drying times, increased mass transfer rates are required [14]. This has been successfully achieved by the SEDS process. This process involves introducing the dry solution and the SF into a particle formation vessel (where temperature and pressure are controlled) through a co-axial nozzle with a mixing chamber. The higher velocity of the SF allows the production of very small sizes, while the mixing of solvent with the SF inside the mixing chamber leads to increased mass transfer of SF into the solvent and vice versa [16]. A high mass transfer allows a faster nucleation and a smaller particle size with agglomeration.
Because of low miscibility of SFCO2 in water, making the use of SEDS quite
challenging for proteins or peptides particle design, this process has been optimised by the use of co-axial three component nozzle. Aqueous solution of protein, organic solvent and SFCO2 are simultaneously introduced to increase the miscibility of SFCO2 in the protein solution.
SEDS is a more controllable and reproducible technique compared with other antisolvent based SF process.
5.4 Precipitation by Compressed Antisolvent
The PCA process is a slight modification of the ASES process. It is a one-step technique used to produce solvent free particles with a narrow size distribution at mild operating conditions [22]. It involves feeding SFCO2 into a precipitator so as to pressurize the precipitator to a desired value. The CO2 flow rate into the precipi- tator is then fixed. The solution of protein is fed into the precipitator through a nozzle. The antisolvent effect of the SFCO2 leads to the precipitation of the protein particles.
Increase in the pressure has been found to help produce a dryer product as the extraction of the liquid solvent to the supercritical phase takes place faster because of a higher solubility of the solvent in the CO2. Furthermore, as pressure increases, the atomisation of the solution produces smaller droplets and the drying time decreases.