key: cord-0072422-mtigvgsl authors: Costa, Clarinda; Casimiro, Teresa; Corvo, Maria Luísa; Aguiar-Ricardo, Ana title: Solid Dosage Forms of Biopharmaceuticals in Drug Delivery Systems Using Sustainable Strategies date: 2021-12-17 journal: Molecules DOI: 10.3390/molecules26247653 sha: 7f2c00011f12f729334abaa8a42e95871712d5a5 doc_id: 72422 cord_uid: mtigvgsl Drug delivery systems (DDS) often comprise biopharmaceuticals in aqueous form, making them susceptible to physical and chemical degradation, and therefore requiring low temperature storage in cold supply and distribution chains. Freeze-drying, spray-drying, and spray-freeze-drying are some of the techniques used to convert biopharmaceuticals-loaded DDS from aqueous to solid dosage forms. However, the risk exists that shear and heat stress during processing may provoke DDS damage and efficacy loss. Supercritical fluids (SCF), specifically, supercritical carbon dioxide (scCO(2)), is a sustainable alternative to common techniques. Due to its moderately critical and tunable properties and thermodynamic behavior, scCO(2) has aroused scientific and industrial interest. Therefore, this article reviews scCO(2)-based techniques used over the year in the production of solid biopharmaceutical dosage forms. Looking particularly at the use of scCO(2) in each of its potential roles—as a solvent, co-solvent, anti-solvent, or co-solute. It ends with a comparison between the compound’s stability using supercritical CO(2)-assisted atomization/spray-drying and conventional drying. Biopharmaceuticals include enzymes, nucleic acids, monoclonal antibodies, and recombinant proteins that are either manufactured or isolated from biological sources for medical application [1] . Due to their greater specificity, affinity, and potency than those of small and chemically synthesized molecules, biopharmaceuticals have aroused tremendous scientific and market interest in recent years. The biopharmaceutical market represented USD 1.6 billion in 2016, increased 102% by 2008 [2] and continues to grow apace. One biopharmaceutical to recently attract attention is recombinant human deoxyribonuclease I (commercially called Dornase alfa) which is widely used in a solution as a cystic fibrosis treatment, as it reduces the viscosity and quantity of airway mucus. Recently, Earhart et al. [3] proposed the use of Dornase alfa for COVID-19 treatment. However, biopharmaceuticals can be challenging to formulate and deliver, owing to their molecular weights that can be up to 500 times that of small molecules. For example, the enzyme golimumab has a molecular weight of 147 kDa whereas quercetin weighs approximately 302 Da, which hinders permeation across biological membranes. It also presents poor biophysical stability, being susceptible to degradation or aggregation depending on the ambient moisture, temperature, or even pH, light, and shear stress [4, 5] . Such challenges can be overcome by associating biopharmaceuticals to drug delivery systems [6] , such as polymers [7] [8] [9] , lipid-based nanoparticles [10] [11] [12] , metal-organic frameworks [5, 13] , and dendrimers [14, 15] . However, biopharmaceutical-based drug delivery systems are usually formulated in aqueous forms [16] which require storage at temperatures between 2 and 8 • C substrate (and the coating agent), without any organic solvents. Upon depressurization to subcritical pressure using a heated nozzle, the substrate precipitates into a lower pressure precipitation chamber as the solution density drops (Figure 1 ) (usually near atmospheric pressure) [35, 38] . The morphology and physicochemical properties of the resulting powders are related to the processing parameters (temperature, pressure, nozzle geometry) and the chemical structure of the material [40] . RESS has been applied mainly to the production of micronized poor water-soluble compounds. In 1993, Reverchon et al. [41] reported the micronization of salicylic acid. The first particles were in needle form, but the work was optimized, and spherical particles resulted. Years later, Yildiz and co-authors [42] reported the micronization of salicylic acid and paclitaxel, using ethanol as a co-solvent to improve salicylic acid's solubility in CO2. This highlights, in fact, one of the technique's limitations. On the one hand, scCO2 presents low solubility for most polymers and pharmaceuticals. On the other, the addition of a second component influences phase behavior. Unfortunately, there is little data available for the multicomponent systems involved in real processes [43] . Years later, a variation in a non-solvent RESS method (RESS-N), introduced by Mishima and co-authors, aroused considerable interest [44] . An organic solvent which does not, in its pure form, solubilize the polymer was added as a co-solvent. Nevertheless, in the presence of those organic solvents, the polymer solubility in CO2 increased significantly. In sum, a suspension of a drug in the ternary system composed by CO2, the non-toxic organic solvent (such as ethanol, isopropanol), and the dissolved polymer was sprayed through the nozzle. The polymer excipient then precipitated around the drug, producing core-shell structured microparticles [45] . Another process modification involved the rapid expansion of a supercritical solution into a liquid solvent (RESOLV) in order to obtain nanoscale particles [46, 47] . In this case, however, the supercritical solution was expanded into a liquid solvent instead of a lower pressure precipitation chamber, and particles with a diameter less than 50 nm were obtained [46] . RESS is more often used with poor water-soluble compound processing, yet it (and its derivatives) has been also applied to encapsulating biopharmaceuticals in drug delivery microsystems. In fact, the processing of water-soluble compounds using RESS can be quite challenging due to CO2′s poor solubility in water. Table 1 displays research reporting on the encapsulation of biopharmaceuticals and the final dry powder properties. The morphology and physicochemical properties of the resulting powders are related to the processing parameters (temperature, pressure, nozzle geometry) and the chemical structure of the material [40] . RESS has been applied mainly to the production of micronized poor water-soluble compounds. In 1993, Reverchon et al. [41] reported the micronization of salicylic acid. The first particles were in needle form, but the work was optimized, and spherical particles resulted. Years later, Yildiz and co-authors [42] reported the micronization of salicylic acid and paclitaxel, using ethanol as a co-solvent to improve salicylic acid's solubility in CO 2 . This highlights, in fact, one of the technique's limitations. On the one hand, scCO 2 presents low solubility for most polymers and pharmaceuticals. On the other, the addition of a second component influences phase behavior. Unfortunately, there is little data available for the multicomponent systems involved in real processes [43] . Years later, a variation in a non-solvent RESS method (RESS-N), introduced by Mishima and co-authors, aroused considerable interest [44] . An organic solvent which does not, in its pure form, solubilize the polymer was added as a co-solvent. Nevertheless, in the presence of those organic solvents, the polymer solubility in CO 2 increased significantly. In sum, a suspension of a drug in the ternary system composed by CO 2 , the non-toxic organic solvent (such as ethanol, isopropanol), and the dissolved polymer was sprayed through the nozzle. The polymer excipient then precipitated around the drug, producing core-shell structured microparticles [45] . Another process modification involved the rapid expansion of a supercritical solution into a liquid solvent (RESOLV) in order to obtain nanoscale particles [46, 47] . In this case, however, the supercritical solution was expanded into a liquid solvent instead of a lower pressure precipitation chamber, and particles with a diameter less than 50 nm were obtained [46] . RESS is more often used with poor water-soluble compound processing, yet it (and its derivatives) has been also applied to encapsulating biopharmaceuticals in drug delivery microsystems. In fact, the processing of water-soluble compounds using RESS can be quite challenging due to CO 2 's poor solubility in water. Table 1 displays research reporting on the encapsulation of biopharmaceuticals and the final dry powder properties. The PGSS technique [51] was introduced in 1994 by Weidner, Knez, and Novak and uses CO 2 as a solute. ScCO 2 is dissolved into a melted material, consisting of drugs, polymers, biopolymers, oils, or fatty acids in a stirred high-pressure vessel until saturation is achieved [33] . The gas saturated liquid is then expanded through the nozzle to an expansion chamber at ambient pressure and lower temperature where the particles precipitate ( Figure 2 ). The PGSS technique [51] was introduced in 1994 by Weidner, Knez, and Novak and uses CO2 as a solute. ScCO2 is dissolved into a melted material, consisting of drugs, polymers, biopolymers, oils, or fatty acids in a stirred high-pressure vessel until saturation is achieved [33] . The gas saturated liquid is then expanded through the nozzle to an expansion chamber at ambient pressure and lower temperature where the particles precipitate ( Figure 2 ). P and T-Pressure and temperature indicator and controller in the autoclave, respectively. PGSS can be more effective than RESS insofar as the compound (drug or polymer) does not need to be dissolved in scCO 2 [52] and no organic solvents are necessary. Moreover, PGSS enables polar and water-soluble compounds to be processed. For this reason, this technique has been widely used in the pharmaceutical field with different compounds. Initially, compounds were processed without carriers. In 1999, Kerč and co-workers [52] used the technique to micronize nifedipine and felodipine (commonly used in hypertension treatment) in order to increase the dissolution rate and their bioavailability. Using temperatures between 150 • C and 185 • C, nifedipine and felodipine microparticles with an irregular shape showed a mean size between 15 and 30 µm and 42 µm, respectively. Smaller microparticles showed an increased dissolution rate. However, the effective surface area was reduced by the drug's hydrophobicity and agglomeration. Thus, to reduce agglomeration, PEG 4000 was added. Microparticles were produced between 50 and 70 • C since the polymer decreased the drug's melting point. It was found that the size reduction and the drug/ PEG 4000 interaction contributed for an enhanced dissolution rate against the pure micronized drugs. Interestingly, this study allowed the further micronization of drug delivery systems with different carriers. Hao et al. [53] produced poly( DL -lactic acid) (P( DL LA)) microparticles, affirming that solid drug particles can be mixed into the plasticized polymer. Figure 3 shows that scCO 2 dissolves into and plasticizes the polymer at temperatures significantly below its glass transition temperature [54] . does not need to be dissolved in scCO2 [52] and no organic solvents are necessary. More over, PGSS enables polar and water-soluble compounds to be processed. For this reason this technique has been widely used in the pharmaceutical field with different com pounds. Initially, compounds were processed without carriers. In 1999, Kerč and co-work ers [52] used the technique to micronize nifedipine and felodipine (commonly used i hypertension treatment) in order to increase the dissolution rate and their bioavailability Using temperatures between 150 °C and 185°C, nifedipine and felodipine microparticle with an irregular shape showed a mean size between 15 and 30 µ m and 42 µ m, respec tively. Smaller microparticles showed an increased dissolution rate. However, the effec tive surface area was reduced by the drug's hydrophobicity and agglomeration. Thus, t reduce agglomeration, PEG4000 was added. Microparticles were produced between 50 an 70 °C since the polymer decreased the drug's melting point. It was found that the siz reduction and the drug/ PEG4000 interaction contributed for an enhanced dissolution rat against the pure micronized drugs. Interestingly, this study allowed the further micron zation of drug delivery systems with different carriers. Hao et al. [53] produced poly(D lactic acid) (P(DLLA)) microparticles, affirming that solid drug particles can be mixed int the plasticized polymer. Figure 3 shows that scCO2 dissolves into and plasticizes the po ymer at temperatures significantly below its glass transition temperature [54] . A year later, Hao et al. [56] applied PGSS to produce PEG microparticles and ob served that the temperature increase and the pressure decrease favored the production o spherical particles. These works contributed to the development of the CriticalMix™solvent-free encapsulation process based on PGSS [57] . Rodrigues et al. [58] reported th encapsulation of theophylline-a bronchodilator-in hydrogenated palm oil, yielding m croparticles sized between 2 and 3 µ m. Higher working pressures (P) were found to lea to a more spherical size and a more limited distribution. Lower P of 120 or 140 bar led t agglomerated structures. Theophylline encapsulation was lower since the drug wa mostly located on the carrier's surface. More recently, Tokunaga et al. [59] reported th encapsulation of an amino acid, phenylalanine, in Eudragit L100. The work was carrie out at 50 °C, between 80 and 120 bar. Microparticles sized from 200 µ m to 140 µ m wer obtained for the respective pressures. At 100 bar, an encapsulation efficiency (EE) of 68.7% was achieved. In fact, the PGSS technique produced microparticles with a wide range o diameters, depending on the working parameters. PGSS can encapsulate biopharmaceuticals at relatively low temperatures, whic makes it attractive for thermolabile compound processing. Moreover, the lack of organ solvents contributes to the integrity of compounds' activity. In 2005, Whitaker et al. [60 reported the encapsulation of ribonuclease A (RNase A), lysozyme, recombinant huma insulin, and salmon calcitonin in P(DLLA) microparticles sized between 10 and 300 µ m Neither enzyme demonstrated an alteration. In turn, insulin activity decreased (althoug A year later, Hao et al. [56] applied PGSS to produce PEG microparticles and observed that the temperature increase and the pressure decrease favored the production of spherical particles. These works contributed to the development of the CriticalMix™-a solvent-free encapsulation process based on PGSS [57] . Rodrigues et al. [58] reported the encapsulation of theophylline-a bronchodilator-in hydrogenated palm oil, yielding microparticles sized between 2 and 3 µm. Higher working pressures (P) were found to lead to a more spherical size and a more limited distribution. Lower P of 120 or 140 bar led to agglomerated structures. Theophylline encapsulation was lower since the drug was mostly located on the carrier's surface. More recently, Tokunaga et al. [59] reported the encapsulation of an amino acid, phenylalanine, in Eudragit L100. The work was carried out at 50 • C, between 80 and 120 bar. Microparticles sized from 200 µm to 140 µm were obtained for the respective pressures. At 100 bar, an encapsulation efficiency (EE) of 68.7% was achieved. In fact, the PGSS technique produced microparticles with a wide range of diameters, depending on the working parameters. PGSS can encapsulate biopharmaceuticals at relatively low temperatures, which makes it attractive for thermolabile compound processing. Moreover, the lack of organic solvents contributes to the integrity of compounds' activity. In 2005, Whitaker et al. [60] reported the encapsulation of ribonuclease A (RNase A), lysozyme, recombinant human insulin, and salmon calcitonin in P( DL LA) microparticles sized between 10 and 300 µm. Neither enzyme demonstrated an alteration. In turn, insulin activity decreased (although less so in the case of in P( DL LA) insulin encapsulation than for the control insulin powder). As for salmon calcitonin activity, the protein concentration was too low to measure. Salmaso et al. [61] describe an improved PGSS technique (GAMA-gas-assisted melting atomization) applied to the micronization of protein-loaded lipid particles. In this case, the protein and the melted lipid were loaded into a melting chamber that was then pressurized with scCO 2 at 150 bar at a working temperature (T) of 40 • C, while undergoing stirring. The loaded lipid particles were dried with a stream of co-axially injected hot air. Vezzù et al. [62] encapsulated RNase A in lipids and polymers and observed that smaller particles were produced at lower temperatures. Table 2 reports the PGSS encapsulation of a range of biopharmaceuticals. In 1997, Sievers et al. [68] proposed a PGSS improvement: the carbon dioxide-assisted nebulization with a bubble dryer (CAN-BD). This technique enables the use of any watersoluble compound, as well as the nebulization of inorganic salts or proteins [69] . Here, CO 2 serves as a co-solute. An aqueous stream containing the drug and any excipients or stabilizers (typically 1 to 10% of the total solids dissolved [33] ) is mixed with supercritical CO 2 in a low-dead-volume mixing tee, forming a gas-liquid emulsion. This is then rapidly decompressed through a flow restrictor, forming an aerosol of droplets containing dissolved CO 2 . Upon expanding the dissolved CO 2 , the droplets rupture, and a fine aerosol, usually of diameters below 5 µm, is produced (Figure 4 ). It is important to point out that the expansion is enhanced since CO 2 is one of the most soluble gases in water (1.6 mole% at 63 • C and 1500 psi). Dry particles are obtained in a furnace [70] , and micronized particles are then collected on a filter. Due to the use of the aqueous stream, CAN-BD has been widely applied to processing water-soluble compounds. In 2001, Sellers et al. [71] reported the encapsulation of proteins (lysozyme and lactate dehydrogenase) using sucrose and mannitol as excipients for pulmonary delivery. A buffer was used to neutralize any potential acidification of the solution upon the addition CO2 to avoid damaging the protein. The proteins were also found to maintain their activity after rehydration due to the excipients and surfactant. Years later, Sievers et al. [72] reported the encapsulation of hepatitis B surface antigen (HBsAg) and a live-attenuated measles virus by trehalose and sucrose. In addition to achieving powder stability after 43 days of storage, they were also able to provide pulmonary administration. In 2008, Cape et al. [73] reported an impressive review of the CAN-BD applied to biopharmaceutical processing. Curiously, since then, CAN-BD has been mostly used to produce vaccines, such as the Edmonston-Zagreb liveattenuated measles virus vaccine [74] [75] [76] , and inhalable antibiotics [77] . To our knowledge, no data on biopharmaceuticals in solid dosage forms have been reported. Due to the use of the aqueous stream, CAN-BD has been widely applied to processing water-soluble compounds. In 2001, Sellers et al. [71] reported the encapsulation of proteins (lysozyme and lactate dehydrogenase) using sucrose and mannitol as excipients for pulmonary delivery. A buffer was used to neutralize any potential acidification of the solution upon the addition CO 2 to avoid damaging the protein. The proteins were also found to maintain their activity after rehydration due to the excipients and surfactant. Years later, Sievers et al. [72] reported the encapsulation of hepatitis B surface antigen (HBsAg) and a live-attenuated measles virus by trehalose and sucrose. In addition to achieving powder stability after 43 days of storage, they were also able to provide pulmonary administration. In 2008, Cape et al. [73] reported an impressive review of the CAN-BD applied to biopharmaceutical processing. Curiously, since then, CAN-BD has been mostly used to produce vaccines, such as the Edmonston-Zagreb live-attenuated measles virus vaccine [74] [75] [76] , and inhalable antibiotics [77] . To our knowledge, no data on biopharmaceuticals in solid dosage forms have been reported. Supercritical assisted atomization (SAA) is based on the PGSS and CAN-BD techniques. First patented by Reverchon in 2001, it consists of the production of nano and microparticles from liquid formulations [78] . This one-step process is very similar to spraydrying but is aided by scCO 2 and it can also be called supercritical CO 2 -assisted spray drying (SASD) [31, 43] . scCO 2 acts as a co-solute and is solubilized into a liquid solution containing the drug and/or a carrier system. The resulting near-equilibrium mixture flowing out of the saturator is atomized using a nozzle in a precipitation chamber at near atmospheric pressure, where it is dried with a hot air/nitrogen flow [43, 79] which serves to avoid the Joule-Thomson effect upon decompression. Two atomization processes take place: (i) droplets at the nozzle are produced by pneumatic atomization; and (ii) CO 2 is quickly released from inside the droplets [80] . Figure 5 illustrates a SAA/SASD process. A series of SAA/SASD studies have been published over the years reporting microparticle production, even for poorly water-soluble compounds. In 2002, Reverchon and co-authors reported the micronization of various compounds using SAA, from salts to corticosteroids [81] . Next, several demonstrated the micronization of antibiotics for inhalation [82] [83] [84] . The research continued, and later polymeric microparticles (chitosan [85] , poly(methyl methacrylate), and poly-L-lactide [86] ) were produced using the technique. Reverchon, in 2007, proposed to produce a composite, in which ampicillin trihydrate and chitosan were selected as a model drug and carrier, respectively, upscaling the technique for the production of a drug delivery system [87] . Several projects have developed drug delivery systems for inhalation. Cabral et al. [88] reported the production of inhalable ibuprofen-loaded chitosan microparticles. Nano-in-microparticles were also developed for effective pulmonary drug delivery using dendrimers [89] and gold nanoparticles [90] as well as gold-coated magnetite nanocomposites [91] . As in the case of RESS, SAA/SASD has also undergone modifications, such as the introduction of a hydrodynamic cavitation mixer (HCM) [92] , to improve mass transfer. Smaller diameter microparticles were reported with a narrower particle size distribution than those produced without HCM. SAA/SASD for encapsulating and delivering biopharmaceuticals (Table 3) has been studied for a large range of compounds from proteins [30] to nucleic acids [93] . Additionally, the encapsulation of a hydrophilic dye as a molecule model (5(6)carboxyfluorescein) has recently been encapsulated in liposomes [94] upon encapsulating trehalose using SASD. The liposomes kept their structure upon resuspension and the encapsulation efficiency was above 95%. The powders showed a mass median aerodynamic diameter of 1.75 ± 0.04 µm and a fine particle fraction of 65 ± 3%, making them fit for pulmonary administration. Storage stability assays at relative humidities of 4%, 50%, and 78% at 20 °C for 7 and 30 days then revealed that the dry powder A series of SAA/SASD studies have been published over the years reporting microparticle production, even for poorly water-soluble compounds. In 2002, Reverchon and co-authors reported the micronization of various compounds using SAA, from salts to corticosteroids [81] . Next, several demonstrated the micronization of antibiotics for inhalation [82] [83] [84] . The research continued, and later polymeric microparticles (chitosan [85] , poly(methyl methacrylate), and poly-L -lactide [86] ) were produced using the technique. Reverchon, in 2007, proposed to produce a composite, in which ampicillin trihydrate and chitosan were selected as a model drug and carrier, respectively, upscaling the technique for the production of a drug delivery system [87] . Several projects have developed drug delivery systems for inhalation. Cabral et al. [88] reported the production of inhalable ibuprofen-loaded chitosan microparticles. Nano-in-microparticles were also developed for effective pulmonary drug delivery using dendrimers [89] and gold nanoparticles [90] as well as gold-coated magnetite nanocomposites [91] . As in the case of RESS, SAA/SASD has also undergone modifications, such as the introduction of a hydrodynamic cavitation mixer (HCM) [92] , to improve mass transfer. Smaller diameter microparticles were reported with a narrower particle size distribution than those produced without HCM. SAA/SASD for encapsulating and delivering biopharmaceuticals (Table 3) has been studied for a large range of compounds from proteins [30] to nucleic acids [93] . Additionally, the encapsulation of a hydrophilic dye as a molecule model (5(6)-carboxyfluorescein) has recently been encapsulated in liposomes [94] upon encapsulating trehalose using SASD. The liposomes kept their structure upon resuspension and the encapsulation efficiency was above 95%. The powders showed a mass median aerodynamic diameter of 1.75 ± 0.04 µm Molecules 2021, 26, 7653 9 of 19 and a fine particle fraction of 65 ± 3%, making them fit for pulmonary administration. Storage stability assays at relative humidities of 4%, 50%, and 78% at 20 • C for 7 and 30 days then revealed that the dry powder formulations kept their liposome structural stability at relative humidities of 4% and 50% at 20 • C for 30 days. [93] An adaptation of SAA to produce liposomes was recently reported [103] . Supercritical assisted Liposome formation (SuperLip) is a continuous operative process, in which biopharmaceuticals can be encapsulated in liposomes, with high reproducibility [104, 105] . It is important to point out that these vesicles are obtained in aqueous bulk instead of solid dosage forms. Ventosa and co-workers [106] reported the depressurization of an expanded liquid organic solution (DELOS) crystallization technique twenty years ago, and it has been commonly used for producing nano-and micro-sized crystalline particles. In this process, at certain temperature and pressure conditions, a compressed gas, e.g., CO 2 , is completely miscible with the organic solvent, acting as a co-solvent, unlike in other techniques [107] . This is a three-step process. First, the solute is dissolved in the organic solvent, below the saturation limit, at working temperature and atmospheric pressure. Then, an expanded liquid mixture is obtained by adding the compressed fluid, at high working pressure, which should not exceed the critical point of the CO 2 /solvent mixture. Finally, depressurization takes place by way of using a non-returning valve upon the rapid reduction of the pressure of the expanded liquid mixture, leading to the precipitation of nano-or microparticles [33, 107] (Figure 6 ). ecules 2021, 26, x FOR PEER REVIEW 11 of commonly used for producing nano-and micro-sized crystalline particles. In this proce at certain temperature and pressure conditions, a compressed gas, e.g., CO2, is complet miscible with the organic solvent, acting as a co-solvent, unlike in other techniques [10 This is a three-step process. First, the solute is dissolved in the organic solvent, below saturation limit, at working temperature and atmospheric pressure. Then, an expand liquid mixture is obtained by adding the compressed fluid, at high working pressu which should not exceed the critical point of the CO2/solvent mixture. Fina depressurization takes place by way of using a non-returning valve upon the rap reduction of the pressure of the expanded liquid mixture, leading to the precipitation nano-or microparticles [33, 107] (Figure 6 ). Figure 6 . The DELOS process, adapted from Ventosa et al. [106] . P, T: Pressure and temperat indicator and controller in the high-pressure vessel and before and after the non-return valve. Since 2001, DELOS has been applied to the micronization of polymers, surfactan [108] and poorly water-soluble compounds such as naproxen and ibuprofen This process uses less CO2; the filtration takes place at atmospheric pressure, and the la of nozzles improves scalability [112] . A technique for developing unilamellar nanovesic based on DELOS was developed that was similar to the above-mentioned SuperLip [11 Depressurization of an expanded liquid organic solution into aqueous solution (DELO susp) is mainly applied to produce nanovesicles to carry nucleic acids and enzym [50, 114, 115] . However, loaded liposomes are obtained in aqueous bulk rather than in dried form. Figure 6 . The DELOS process, adapted from Ventosa et al. [106] . P, T: Pressure and temperature indicator and controller in the high-pressure vessel and before and after the non-return valve. Since 2001, DELOS has been applied to the micronization of polymers, surfactants, [108] and poorly water-soluble compounds such as naproxen and ibuprofen [109] [110] [111] . This process uses less CO 2 ; the filtration takes place at atmospheric pressure, and the lack of nozzles improves scalability [112] . A technique for developing unilamellar nanovesicles based on DELOS was developed that was similar to the above-mentioned SuperLip [113] . Depressurization of an expanded liquid organic solution into aqueous solution (DELOS-susp) is mainly applied to produce nanovesicles to carry nucleic acids and enzymes [50, 114, 115] . However, loaded liposomes are obtained in aqueous bulk rather than in the dried form. Since scCO 2 is a poor solvent for most pharmaceutical compounds and polymers, some approaches use it as an anti-solvent. In a supercritical anti-solvent (SAS), a solute is dissolved into an organic solvent and precipitates when scCO 2 is added. In fact, the precipitation occurs by the diffusion of CO 2 in the organic solvent, expanding the liquid mixture, decreasing its density, and consequently, its solvation power, until nucleation and precipitation occur [116] . The mass transfer between the CO 2 and the organic solution decreases the surface tension, which is strong enough to control droplet shape [117] . Figure 7 illustrates this process. Molecules 2021, 26, x FOR PEER REVIEW Figure 7 . The SAS process, adapted from Aguiar-Ricardo et al. [39] . P, T: Pressure and tem indicator and controller in the precipitation chamber, respectively. The CO2 is pumped until a desired pressure in the heated precipitator is ac The organic solvent with the solute(s) is then injected by nozzle. In the ca drug/polymer system, both precipitate under supersaturation and the drug are t into the polymer matrix [118] . The loaded polymer precipitates on a filter and the solvent/CO2 is recovered for further separation [119] . Impressive reviews rel pharmaceutical encapsulation using the SAS and SAS-based methods hav published [117, 119] . However, the use of organic solvents to dissolve the solu represent an obstacle to biopharmaceutical applications [118] . Even so, some resea reported successful biopharmaceutical drying with the SAS technique (Table 4 ). I Yeo et al. [120] reported the first production of microparticulate insulin powder SAS, obtaining powders with diameters mostly less than 4 µm. The blood gluco decreased with the administration of the insulin microparticles. Further results s that SAS can compromise proteins' secondary structure, although precipitated recovered its native structure upon redissolution in aqueous media [121] modifications of the SAS process have been proposed to overcome this drawback CO2 also acts as an anti-solvent. However, this review reported little focused atten this aerosol solvent extraction system (ASES) [122] using solution-enhanced disper supercritical fluids (SEDS) [123] , precipitation with a compressed fluid anti-solven [124] and SCF-assisted extraction of emulsions (SFEE) [119, 125] . The CO 2 is pumped until a desired pressure in the heated precipitator is achieved. The organic solvent with the solute(s) is then injected by nozzle. In the case of a drug/polymer system, both precipitate under supersaturation and the drug are trapped into the polymer matrix [118] . The loaded polymer precipitates on a filter and the organic solvent/CO 2 is recovered for further separation [119] . Impressive reviews related to pharmaceutical encapsulation using the SAS and SAS-based methods have been published [117, 119] . However, the use of organic solvents to dissolve the solute may represent an obstacle to biopharmaceutical applications [118] . Even so, some research has reported successful biopharmaceutical drying with the SAS technique (Table 4 ). In 1993, Yeo et al. [120] reported the first production of microparticulate insulin powders using SAS, obtaining powders with diameters mostly less than 4 µm. The blood glucose level decreased with the administration of the insulin microparticles. Further results showed that SAS can compromise proteins' secondary structure, although precipitated insulin recovered its native structure upon redissolution in aqueous media [121] . Some modifications of the SAS process have been proposed to overcome this drawback, where CO 2 also acts as an anti-solvent. However, this review reported little focused attention on this aerosol solvent extraction system (ASES) [122] using solution-enhanced dispersion by supercritical fluids (SEDS) [123] , precipitation with a compressed fluid anti-solvent (PCA) [124] and SCF-assisted extraction of emulsions (SFEE) [119, 125] . [129] 1 N-N-Dimethylformamide; 2 Dichloromethane; 3 Poly-( L -lactide); 4 Hydroxypropyl methyl cellulose phthalate. Biopharmaceutical stability after processing is an interesting drying issue. Despite the advantages of freeze-drying, including the freedom from the need for high temperatures and moisture control, biopharmaceuticals are submitted to different stresses, such as crystallization, dehydration changes, ionic strength changes, interfacial stress, and ice crystal formation [24] . In fact, the drying of enzymes and proteins without excipients can lead to dimers and aggregates [130] . Zhai et al. [131] reported a herpes simplex virus 2 activity recovery of 22% for a minimum number of excipients, whereas a higher recovery (80%) was found when higher percentages of excipients were used. However, recent studies have shown that the effectiveness of the excipients on biopharmaceutical stabilization depends mainly on the protein/enzyme structure [132] . For this reason, the solute concentration and solute-excipient-buffer interaction should be considered for a stable biopharmaceutical solid dosage form, using freeze-drying. Spray-drying is an attractive technique for biopharmaceuticals insofar as it is simple and easy to scale-up, plus it offers particle size control and good aerosolization. However, the biopharmaceutical is subject to thermal, atomization, interfacial, and mechanical stresses [24] . Ajmera et al. [133] reported that the combination of two amino acids-arginine and glycine-was mandatory for catalase stability during spray-drying. Without excipients, the catalase lost around 50% of its activity due to the destruction of hydrophobic interactions during drying and the breakup of intermolecular hydrogen bonds upon removing the hydration shell. With the presence of the amino acids, catalase activity increases to 60-80%. A recent study from Fernandes et al. [134] showed a SOD (Cu, Zn-superoxide dismutase) activity retention between 50-80%, with no loss of SOD conformational stability. They observed, as in the case of freeze-drying, a stabilizing effect induced by the presence of excipients (trehalose and leucine). Ji et al. [135] showed a total lysozyme bioactivity of approximately 71.5% without excipients. A notable increase in bioactivity was observed upon the addition of trehalose, Tween 20, or phosphate-buffered saline (PBS), and at a lower inlet temperature (80 • C). Biopharmaceuticals processed with SAA/SASD are also submitted to atomization stress. However, several studies have shown that protein stability suffers only a slight change during the process. The tunable properties of scCO 2 mean that particle engineering can speed up the process. Du et al. [99] found that an increase of P CO2 led to a loss of relative activity, due to the acidity of CO 2 , with a consequent pH change that provoked protein denaturation. The authors also observed a relative activity of 95% when the mixer was at 100 • C. It is important to call attention to the fact that no excipients were used during drying. Another study from Shen et al. [101] showed the processing of trypsin in SAA-HCM. Free trypsin, without excipients, presented a relative activity of 74.3%, whereas the relative activity of trypsin from spray-drying was 66.1%. Silva et al. [93] also reported a stable nucleic acid after SASD. It would be interesting to study SOD activity from the solid dosage SASD forms to compare with those from freeze-drying and spray-drying. All of these studies show that SAA/SASD is not merely an effective process for particle morphology control and scalability. Processing in mild conditions might also reinforce biopharmaceutical stability without compromising its conformational structure. This work reviews scCO 2 -based techniques for producing solid dosage forms of biopharmaceuticals. scCO 2 provides green and sustainable alternatives to conventional techniques, such as spray-drying for producing dry powders, as it reduces the heat and shear stress and, even when using organic solvents, their amount is lower. The encapsulation of biopharmaceuticals in solid dosage forms using RESS, PGSS, CAN-BD, SAA/SASD, DELOS, and SAS has been intensively reviewed. RESS showed good size control. However, the poor solubility of drugs and polymers in scCO 2 hindered particle recovery leading to scale-up limitations and other disadvantages. In contrast, PGSS presents easy scalability, requires no organic solvents, and can take place at low temperatures. However, since it requires the solubilization of scCO 2 in the melted compound, the technique is limited to pharmaceutical compounds in which CO 2 is highly soluble. Moreover, the fact that the solute must be melted represents a potential issue for heat-sensitive materials. CAN-BD is an interesting method as a wide range of compounds can be processed, although high temperatures are required. SAA/SASD presents good particle size control, high drying and atomization efficiency, and easy tunability to produce microparticles. Yet, small amounts of organic solvents might be required to improve the drying process. Using the DELOS technique, low pressures can be applied for optimum precipitation. However, the process is not applied to biopharmaceuticals (probably since biopharmaceutical crystallization might occur). Finally, SAS was found to be attractive for processing insoluble compounds in CO 2 (as is the case for most drugs). However, the use of organic solvents to dissolve the solute may represent an obstacle to biopharmaceutical applications. Unfortunately, throughout the literature, there is less data about producing solid dosage forms of biopharmaceuticals than that looking into the same system for poorly water-soluble and low molecular weight molecules, excepting SAA/SASD. As a matter of fact, even scCO 2 techniques present impressive and potential results; biopharmaceuticals are very sensitive to their environment and their processing may prove more challenging. For this reason, future research into the development of nano-in-microparticles solid dosage forms is predicted to increase. Biopharmaceuticals are first encapsulated in a nanosystem that is then encapsulated in microparticles. We believe that this scCO 2 -based technique will potentially produce loaded solid dosage forms, without compromising the biopharmaceutical structure or stability. Nanocarrier-Mediated Cytosolic Delivery of Biopharmaceuticals Progress in Biopharmaceutical Development Consideration of Dornase Alfa for the Treatment of Severe Acute Respiratory Distress Syndrome. New Microbes New Infect. 2020 Overcoming the Challenges in Administering Biopharmaceuticals: Formulation and Delivery Strategies Antibodies@MOFs: An In Vitro Protective Coating for Preparation and Storage of Biopharmaceuticals Therapeutic SiRNA: State of the Art Synthesis of Poly(Acyclic Orthoester)s: Acid-Sensitive Biomaterials for Enhancing Immune Responses of Protein Vaccine Incorporation of Lysozyme into a Mucoadhesive Electrospun Patch for Rapid Protein Delivery to the Oral Mucosa Protein-Responsive Protein Release of Supramolecular/Polymer Hydrogel Composite Integrating Enzyme Activation Systems One-Step Microfluidics Production of Enzyme-Loaded Liposomes for the Treatment of Inflammatory Diseases All-in-One Microfluidic Assembly of Insulin-Loaded pH-Responsive Nano-in-Microparticles for Oral Insulin Delivery Liver-Targeted SiRNA Lipid Nanoparticles Treat Hepatic Cirrhosis by Dual Antifibrotic and Anti-Inflammatory Activities Metal-Organic Frameworks as Protective Matrices for Peptide Therapeutics Silencing of SARS-CoV-2 with Modified SiRNA-Peptide Dendrimer Formulation Treatment of acute lung inflammation by pulmonary delivery of anti-TNF-α siRNA with PAMAM dendrimers in a murine model Spray-Drying of Protein Pharmaceuticals: Literature Analysis of Trends in Formulation and Process Attributes. Dry Technol. 2021 SARS-CoV-2 Vaccines in Development Drying Technologies for Biopharmaceutical Applications: Recent Developments and Future Direction Supercritical Carbon Dioxide Design Strategies: From Drug Carriers to Soft Killers Sustainable Strategies for Nano-in-Micro Particle Engineering for Pulmonary Delivery Aerosolizable SiRNA-Encapsulated Solid Lipid Nanoparticles Prepared by Thin-Film Freeze-Drying for Potential Pulmonary Delivery Intravenous Administration of Superoxide Dismutase Entrapped in Long Circulating Liposomes II. In Vivo Fate in a Rat Model of Adjuvant Arthritis Editorial: Freeze-Drying and Process Analytical Technology for Drying Technologies for the Stability and Inhaled Insulin (Exubera ® ): Combining Efficacy and Convenience Why Is Exubera Being Withdrawn? Development of Inhalable Dry Powder Formulation of Basic Fibroblast Growth Factor On the Solubility of Solids in Gases Stabilization of Proteins in Dry Powder Formulations Using Supercritical Fluid Technology Development of PLGA Dry Powder Microparticles by Supercritical CO 2 -Assisted Spray-Drying for Potential Vaccine Delivery to the Lungs Building Dry Powder Formulations Using Supercritical CO 2 Spray Drying Supercritical Assisted Atomization: A Novel Technology for Microparticles Preparation of an Asthma-Controlling Drug Dense CO 2 as a Solute, Co-Solute or Co-Solvent in Particle Formation Processes: A Review Green Chemistry: Principles and Practice Production of Powders and Films by the Rapid Expansion of Supercritical Solutions Rapid Expansion of Supercritical Fluid Solutions: Solute Formation of Powders, Thin Films, and Fibers Rapid Precipitation of Low Vapor Pressure Solids from Supercritical Fluid Solutions: The Formation of Thin Films and Powders Nanomaterials and Supercritical Fluids Supercritical Fluid Manufacture Solubility in Supercritical Carbon Dioxide Salicylic Acid Solubilization in Supercritical CO 2 and Its Micronization by RESS Micronization of Salicylic Acid and Taxol (Paclitaxel) by Rapid Expansion of Supercritical Fluids (RESS) Optimization of Supercritical CO 2 -Assisted Atomization: Phase Behavior and Design of Experiments Microencapsulation of Proteins by Rapid Expansion of Supercritical Solution with a Nonsolvent Formation of Microcapsules of Medicines by the Rapid Expansion of a Supercritical Solution with a Nonsolvent Nanosizing Drug Particles in Supercritical Fluid Processing Solubility Measurement and RESOLV-Assisted Nanonization of Gambogic Acid in Supercritical Carbon Dioxide for Cancer Therapy Microencapsulation of Protein Particles within Lipids Using a Novel Supercritical Fluid Process Microencapsulation of Coenzyme Q 10 in Poly(Ethylene Glycol) and Poly(Lactic Acid) with Supercritical Carbon Dioxide Nanovesicles and Its Use for Nucleic Acid Delivery Process for Preparing Particles or Powders. WO Patent Micronization of Drugs Using Supercritical Carbon Dioxide Plasticization and Spraying of Poly (DL-lactic Acid) Using Supercritical Carbon Dioxide: Control of Particle Size Interaction of Supercritical Carbon Dioxide with Polymers. I. Crystalline Polymers Evaluation of P(L)LA-PEG-P(L)LA as Processing Aid for Biodegradable Particles from Gas Saturated Solutions (PGSS) Process Supercritical Fluid Assisted Melting of Poly(Ethylene Glycol): A New Solvent-Free Route to Microparticles Sustained Release HGH Microsphere Formulation Produced by a Novel Supercritical Fluid Technology: In Vivo Studies Microcomposites Theophylline/Hydrogenated Palm Oil from a PGSS Process for Controlled Drug Delivery Systems Microencapsulation of Drug with Enteric Polymer Eudragit L100 for Controlled Release Using the Particles from Gas Saturated Solutions (PGSS) Process The Production of Protein-Loaded Microparticles by Supercritical Fluid Enhanced Mixing and Spraying Biopharmaceutical Characterisation of Insulin and Recombinant Human Growth Hormone Loaded Lipid Submicron Particles Produced by Supercritical Gas Micro-Atomisation Production of Lipid Microparticles Containing Bioactive Molecules Functionalized with PEG Production of Solid Lipid Submicron Particles for Protein Delivery Using a Novel Supercritical Gas-Assisted Melting Atomization Process Coenzyme Q10 Nanoparticles Prepared by a Supercritical Fluid-Based Method Stability of Human Growth Hormone in Supercritical Carbon Dioxide Preparation and Characterization of Progesterone Dispersions Using Supercritical Carbon Dioxide Encapsulation of Vitamin B2 in Solid Lipid Nanoparticles Using Supercritical CO 2 Methods for Fine Particle Formation Formation of Aqueous Small Droplet Aerosols Assisted by Supercritical Carbon Dioxide Submicron-Sized Spherical Yttrium Oxide Based Phosphors Prepared by Supercritical CO 2 -Assisted Aerosolization and Pyrolysis Dry Powders of Stable Protein Formulations from Aqueous Solutions Prepared Using Supercritical CO 2 -assisted Aerosolization Near-Critical Fluid Micronization of Stabilized Vaccines, Antibiotics and Anti-Virals Preparation of Active Proteins, Vaccines and Pharmaceuticals as Fine Powders Using Supercritical or Near-Critical Fluids Dry Powder Measles Vaccine: Particle Deposition, Virus Replication, and Immune Response in Cotton Rats Following Inhalation Stabilizing Formulations for Inhalable Powders of Live-Attenuated Measles Virus Vaccine Successful Respiratory Immunization with Dry Powder Live-Attenuated Measles Virus Vaccine in Rhesus Macaques Inhalable Antibiotics Manufactured Through Use of Near-Critical or Supercritical Fluids Process For The Production Of Micro And/Or Nano Particles Cabral-Marques, H. Preparation of Ibuprofen/Hydroxypropyl-γ-Cyclodextrin Inclusion Complexes Using Supercritical CO 2 -Assisted Spray Drying Micronization of Lysozyme by Supercritical Assisted Atomization Supercritical-Assisted Atomization To Produce Micro-and/or Nanoparticles of Controlled Size and Distribution Ampicillin Micronization by Supercritical Assisted Atomization Della Micronization of Antibiotics by Supercritical Assisted Atomization Erythromycin Micro-Particles Produced by Supercritical Fluid Atomization Chitosan Microparticles Production by Supercritical Fluid Processing Polymer Microparticles Production by Supercritical Assisted Atomization Drug-Polymer Microparticles Produced by Supercritical Assisted Atomization Design of Experiments Approach on the Preparation of Dry Inhaler Chitosan Composite Formulations by Supercritical CO 2 -Assisted Spray-Drying Nano-in-Micro POxylated Polyurea Dendrimers and Chitosan Dry Powder Formulations for Pulmonary Delivery Aerosolizable Gold Nano-in-Micro Dry Powder Formulations for Theragnosis and Lung Delivery Supercritical CO 2 -Assisted Spray Drying of Strawberry-Like Gold-Coated Magnetite Nanocomposites in Chitosan Powders for Inhalation Supercritical Fluid Assisted Atomization Introduced by Hydrodynamic Cavitation Mixer (SAA-HCM) for Micronization of Levofloxacin Hydrochloride Rational Design of Multistage Drug Delivery Vehicles for Pulmonary RNA Interference Therapy Inhalable Hydrophilic Molecule-Loaded Liposomal Dry Powder Formulations Using Supercritical CO 2 -Assisted Spray-Drying Production of New Haemostatic Materials by Deposition of Dispersed Proteins onto Porous Matrices Using Supercritical Carbon Dioxide. Russ Albumin/Gentamicin Microspheres Produced by Supercritical Assisted Atomization: Optimization of Size, Drug Loading and Release Controllable Preparation and Formation Mechanism of BSA Microparticles Using Supercritical Assisted Atomization with an Enhanced Mixer A New Supercritical Assisted Atomization Configuration, for the Micronization of Thermolabile Compounds Supercritical Fluid Assisted Atomization Introduced by an Enhanced Mixer for Micronization of Lysozyme: Particle Morphology, Size and Protein Stability Bioactive Insulin Microparticles Produced by Supercritical Fluid Assisted Atomization with an Enhanced Mixer Supercritical Fluid Assisted Production of Micrometric Powders of the Labile Trypsin and Chitosan/Trypsin Composite Microparticles Preparation of Micrometric Powders of Parathyroid Hormone (PTH1-34)-Loaded Chitosan Oligosaccharide by Supercritical Fluid Assisted Atomization Liposomes Preparation Using a Supercritical Fluid Assisted Continuous Process Efficient Encapsulation of Proteins in Submicro Liposomes Using a Supercritical Fluid Assisted Continuous Process A Versatile Supercritical Assisted Process for the One-Shot Production of Liposomes Depressurization of an Expanded Liquid Organic Solution (DELOS): A New Procedure for Obtaining Submicron-Or Micron-Sized Crystalline Particles A Crystallization Technique Using Compressed Fluids-1. Comparison to the GAS Crystallization Method Synergistic Solubility Behaviour of a Polyoxyalkylene Block Co-Polymer and Its Precipitation from Liquid CO2-Expanded Ethanol as Solid Microparticles Solubility Behaviors of Ibuprofen and Naproxen Drugs in Liquid "CO 2 -Organic Solvent" Mixtures Crystallization of Microparticulate Pure Polymorphs of Active Pharmaceutical Ingredients Using CO 2 -Expanded Solvents Influence of the Preparation Route on the Supramolecular Organization of Lipids in a Vesicular System Nanostructuring Molecular Materials as Particles and Vesicles for Drug Delivery, Using Compressed and Supercritical Fluids Preparation of Uniform Rich Cholesterol Unilamellar Nanovesicles Using CO 2 -Expanded Solvents Application of Quality by Design to the Robust Preparation of a Liposomal GLA Formulation by DELOS-Susp Method Multifunctional Nanovesicle-Bioactive Conjugates Prepared by a One-Step Scalable Method Using CO 2 -Expanded Solvents Applications of Supercritical CO 2 in the Fabrication of Polymer Systems for Drug Delivery and Tissue Engineering Application of Supercritical Antisolvent Method in Drug Encapsulation: A Review Production of Protein-Loaded Polymeric Microcapsules by Compressed CO 2 in a Mixed Solvent Supercritical Antisolvent Process for Pharmaceutical Applications: A Review. Processes 2020, 8, 938 Formation of Microparticulate Protein Powder Using a Supercritical Fluid Antisolvent Secondary Structure Characterization of Microparticulate Insulin Powders Influence of Gas Density and Pressure on Microparticles Produced with the ASES Process Supercritical Fluid Processing of Materials from Aqueous Solutions: The Application of SEDS to Lactose as a Model Substance Encapsulation of Lysozyme in a Biodegradable Polymer by Precipitation with a Vapor-over-Liquid Antisolvent Supercritical Fluid Technology: An Emphasis on Drug Delivery and Related Biomedical Applications Anti-Solvent Effect in the Production of Lysozyme Nanoparticles by Supercritical Fluid-Assisted Atomization Processes The Preparation of BSA-PLLA Microparticles in a Batch Supercritical Anti-Solvent Process Compressed CO 2 Antisolvent Precipitation of Lysozyme Preparation of Hydroxypropyl Methyl Cellulose Phthalate Nanoparticles with Mixed Solvent Using Supercritical Antisolvent Process and Its Application in Co-Precipitation of Insulin Freeze-Drying of Proteins: Some Emerging Concerns Effect of Freezing Rates and Excipients on the Infectivity of a LiveViral Vaccine during Lyophilization The Preservation of Lyophilized Human Growth Hormone Activity: How Do Buffers and Sugars Interact? Stabilisation of Proteins via Mixtures of Amino Acids during Spray Drying Dry Powder Inhaler Formulation of Cu,Zn-Superoxide Dismutase by Spray Drying: A Proof-of-Concept Investigation of Factors Affecting the Stability of Lysozyme Spray Dried from Ethanol-Water Solutions The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.