J. of Supercritical Fluids 94 (2014) 189–197 Contents lists available at ScienceDirect The Journal of Supercritical Fluids j our na l ho me page: www.elsevier.com/locate/supflu Antisolvent micronization of BSA using supercritical mixtures carbon dioxide + organic solvent Valentina Prosapio, Ernesto Reverchon, Iolanda De Marco ∗ Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy a r t i c l e i n f o Article history: Received 20 May 2014 Received in revised form 17 July 2014 Accepted 17 July 2014 Available online 27 July 2014 Keywords: Expanded liquid antisolvent process Hydrosoluble proteins Bovine serum albumin Supercritical fluids a b s t r a c t In this work, expanded liquid antisolvent (ELAS) process has been used to micronize bovine serum albu- min (BSA) solubilized in water. Carbon dioxide mixtures with ethanol, acetone or isopropyl alcohol, at expanded liquid conditions, have been used as the antisolvent. The effect of process parameters, such as the kind of co-antisolvent and the organic co-antisolvent/water/carbon dioxide mole fraction on the morphology and dimensions of the precipitates, was studied. Changing co-antisolvent and operating con- ditions, we obtained nanoparticles (with a mean diameter of about 60 nm ± 10 nm), sub-microparticles (with a mean diameter of 470 nm ± 130 nm), microparticles (with a mean diameter of 0.93 m ± 0.37 m) and expanded microparticles with an empty core (with a mean diameter of about 9 m ± 5 m). Fourier transform infrared analysis on BSA powders revealed that, using acetone as co-antisolvent, no modifica- tions of the protein secondary structure were induced by ELAS processing. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Micronization of high-added value products, such as pharma- ceuticals, nutraceuticals, proteins and enzymes, has been fre- quently attempted using supercritical carbon dioxide (SC-CO 2 ) based techniques [1–4]. Among them, supercritical antisolvent (SAS) has been successfully used to obtain microparticles and nanoparticles of various materials, changing the organic solvent and the operating conditions [1,5]. Studies on precipitation mech- anisms and attempts at the modeling of SAS process were also performed [6–10]. SAS is based on two pre-requisites: (a) organic solvent and SC-CO 2 have to be perfectly miscible at the process conditions; (b) the solute to be micronized has to be soluble in the organic solvent and has to possess a near zero solubility in the mixture formed by SC-CO 2 and the organic solvent. It has been demonstrated that, for standard SAS processing, thermodynamic conditions of complete miscibility represent the necessary condition for successful micronization; whereas, fluid dynamics and mass transfer concur in determining the morphology of particles produced [7,8]. The condition of miscibility, between solvent and antisolvent, limits the applicability of the SAS process to hydrophobic com- pounds that are generally soluble in SC-CO 2 . Water solubility in ∗ Corresponding author. Tel.: +39 089 964066; fax: +39 89 964057. E-mail address: idemarco@unisa.it (I. De Marco). carbon dioxide is, instead, very limited at the usual SAS condi- tions (40–60 ◦ C and 100–250 bar) [11]. Since several categories of pharmaceutical products, as, for example, proteins and enzymes, are water-soluble, a modification of the SAS process is required to extend its applicability to water soluble compounds. An interesting opportunity could be to add to the supercritical antisolvent an organic solvent, completely miscible with SC-CO 2 at process conditions, to form a supercritical mixture in which water solubility is largely enhanced or complete. Practically speaking, the mixture CO 2 plus organic solvent can act as a hydrophilic antisol- vent in the modified SAS process. Some papers can be found in the literature, in which CO 2 + organic solvent were used in the attempt of processing hydrophilic compounds by supercritical antisolvent precipitation [12–15]. Some proteins, like, for example, lysozyme, albumin, insulin, recombinant human deoxyribonuclease (rhD- Nase), lysozyme-lactose and rhDNase-lactose were processed by Bustami et al. [12,13] using a 0.2 mole fraction of ethanol or of ethanol and triethylamine in SC-CO 2 as the antisolvent. Irregular and aggregated micrometric particles (3–18 m) were obtained. Bouchard et al. [14,15], processed lysozyme, -glycine and phenylalanine anhydrate, obtaining microparticles in the range 1–50 m using methanol, ethanol, 2-propanol or acetone as co- antisolvent. However, these authors [14,15] processed very small quantities of water solutions (around 0.06 water mole fraction), producing, as a result, extremely small quantities of precipitates. Recently, De Marco and Reverchon [16] proposed the supercrit- ical antisolvent processing of bovine serum albumin (BSA) using http://dx.doi.org/10.1016/j.supflu.2014.07.012 0896-8446/© 2014 Elsevier B.V. All rights reserved.