Extraction and purification of violacein from Yarrowia lipolytica cells using aqueous solutions of surfactants

BACKGROUND: L-Asparaginase (ASNase) is an important biopharmaceutical for the treatment of acute lymphoblastic leukemia (ALL); however, with some restrictions due to its high manufacturing costs. Aqueous biphasic systems (ABS) have been suggested as more economical platforms for the separation/purification of proteins, but a full understanding of the mechanisms behind the ASNase partition is still a major challenge. Polymer/salt-based ABS with different driving-forces (salting-out and hydrophilicity/hydrophobicity effects) were herein applied to control the partition of commercial ASNase. RESULTS: The main results showed the ASNase partition to the salt- or polymer-rich phase depending on the ABS studied, with extraction efficiencies higher than 95%. For systems composed of inorganic salts, the ASNase partition was controlled by the polyethylene glycol (PEG) molecular weight used. Cholinium-salts-based ABS were able to promote a preferential ASNase partition to the polymer-rich phase using PEG-600 and to the salt-rich phase using a more hydrophobic polypropylene glycol (PPG)-400 polymer. It was possible to select the ABS composed of PEG-2000 + potassium phosphate buffer as the most efficient to separate the ASNase from the main contaminant proteins (purification factor = 2.4 ± 0.2), while it was able to maintain the enzyme activity for posterior application as part of a therapeutic. CONCLUSION: Polymer/salt ABS can be used to control the partition of ASNase and adjust its purification yields, demonstrating the ABS potential as more economic platform for the selective recovery of therapeutic enzymes from complex broths.publishe

Imidazolium-based ionic liquids as adjuvants to form Polyethylene Glycol with Salt Buffer Aqueous Biphasic Systems

Aqueous biphasic systems (ABS) are biocompatible systems applied in the extraction of biomolecules. Despite the biocompatibility of polymers and, particularly polyethylene glycol (PEG), to form ABS, their limitation in terms of phase separation is recognized. A new approach was recently proposed based on the use of ionic liquids (ILs) as adjuvants in ABS, enlarging the polarity range of these systems. Up to now, the effects of ILs in PEG-salt ABS have been poorly described. To overcome this limitation, the phase diagrams of imidazolium-based ILs acting as adjuvants in ABS based in PEG with potassium salt buffers (pH = 7), that is potassium citrate (C6H5K3O7/C6H8O7) and potassium phosphate (K2HPO4/KH2PO4) buffers, are herein addressed. Imidazolium-based ILs were focused in this work, since they have been applied on the purification of several biomolecules with success, even as adjuvants or electrolytes. The phase diagrams were mapped out for PEG/salt ABS without adjuvants. In this work, systems composed of PEG (1000, 1500, 2000, 3350, 4000, 6000, and 8000) with potassium phosphate buffer and PEG (2000, 6000, 10 000, and 20 000) with potassium citrate buffer were tested. Moreover, the presence of 5 wt % of imidazolium-based ILs (varying the anion moiety) for the system PEG 1500 with potassium phosphate buffer was also investigated. Imidazolium-based ILs with different anions were tested to investigate a large range of polarities attributed to the adjuvant. Moreover, the effect of the adjuvant content (5, 10, and 20 wt %) in the PEG 2000 with potassium citrate buffer system was studied for two distinct ILs, namely [C4mim][CF3SO3] and [C4mim][(CH3O)2PO2], with lower and higher energy of intramolecular hydrogen bond, EHB, respectively, a parameter representing the ions' hydration. A correlation between the anion moiety of imidazolium-based IL and the ability to form two phases was observed, being this related to the ILs' anion EHB value. The concentration of the adjuvant confirmed the effects of enhancing or decreasing the ability to form two phases for ILs with lower and higher EHB value, respectively.publishe

Development and characterization of miltefosine-loaded polymeric micelles for cancer treatment

Miltefosine presents antineoplastic activity but high hemolytic potential. Its use in cancer has been limited to treating cutaneous metastasis of breast cancer. To decrease hemolytic potential, we developed a formulation of miltefosine-loaded polymeric micelles (PM) of the copolymer Pluronic-F127. A central composite design was applied and the analysis of variance showed that the optimum level of hydrodynamic diameter and polydispersity index predicted by the model and experimentally confirmed were 29 nm and 0.105, respectively. Thermal analyses confirmed that miltefosine was molecularly dispersed within PM. Pluronic-F127 PM with miltefosine 80 μM presented a significant reduction of hemolytic effect (80%, p < 0.05) in comparison to free drug. In vitro assays against HeLa carcinoma cells demonstrated similar cytotoxicity to free miltefosine and PM. Our results suggest that, by lowering hemolytic potential, miltefosine-loaded Pluronic-F127 PM a promising alternative to broaden this drug use in cancer therapy, as well as of other alkylphosphocholines.Fil: Valenzuela Oses, Johanna K.. Universidade de Sao Paulo; BrasilFil: García, Mónica Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; ArgentinaFil: Feitosa, Valker A.. Universidade de Sao Paulo; Brasil. Institute for Technological Research; BrasilFil: Pachioni Vasconcelos, Juliana A.. Universidade de Sao Paulo; BrasilFil: Gomes-Filho, Sandro M.. Universidade de Sao Paulo; BrasilFil: Lourenço, Felipe R.. Universidade de Sao Paulo; BrasilFil: Cerize, Natalia N.P.. Institute for Technological Research; BrasilFil: Bassères, Daniela S.. Universidade de Sao Paulo; BrasilFil: Rangel-Yagui, Carlota O.. Universidade de Sao Paulo; Brasi

An integrated process combining the reaction and purification of PEGylated proteins

A downstream process combining PEGylation reaction and the use of enzyme conjugates acting as phase-forming components of aqueous biphasic systems (ABS) is proposed here. In this approach, citrate buffer (pH = 7.0) was used simultaneously to stop the reaction (avoiding the use of hydroxylamine) and as a phase forming agent inducing the phase separation of the PEGylated proteins. The partition of the bioconjugates was assessed using two model enzymes of small size [cytochrome c (Cyt-c) and lysozyme (LYS)], and two of large size [l-asparaginase (ASNase) and catalase (CAT)] as well as reactive PEG of 5, 10, 20 and 40 kDa. The effect of the reaction time on the PEGylation and recovery steps was also evaluated. All reactive PEGs allowed high selectivity in the separation of PEGylated proteins from native proteins (S > 100). A positive effect in terms of selectivity was found for longer reaction times. It allowed greater amounts of PEGylated proteins, with an increase of the PEG-protein rich-phase volume (top phase), allowing 100% of recovery of PEGylated proteins. More selective systems were obtained for Cyt-c and LYS (S > 100) compared to those for ASNase and CAT (40 < S < 60); nevertheless for all, the native and PEGylated proteins had their biological activity preserved. Envisioning the industrial potential evaluation, an integrated process diagram was defined combining the PEGylation reaction with the purification of the protein conjugates. Two different scenarios were investigated considering the PEGylation reaction performance. For both approaches (complete and incomplete PEGylation reaction), high recovery yields and purities were achieved for the PEGylated conjugates (92.1 ± 0.4% < %RecTCyt-c-PEG < 98.1 ± 0.1%; 84.6% < purity < 100%) and for the unreacted enzyme (%RecBCyt-c = 81 ± 1%; purity = 97.7%), while maintaining their structural integrity.publishe

Continuous separation of cytochrome-c PEGylated conjugates by fast centrifugal partition chromatography

Herein, the effective use of aqueous biphasic systems (ABS) in Fast Centrifugal Partition Chromatography (FCPC) for the purification of PEGylated cytochrome c conjugates is shown. High recoveries (between 88% and 100%) and purities (∼100%) were obtained. Both the unreacted cytochrome c and solvents may be recovered and reused, thus allowing the design of a sustainable process in a continuous regime for the isolation of bioconjugates. This process allowed the reduction of the complete E-factor and carbon footprint at circa 100% and 67%, respectively, reinforcing the important environmental contribution of recycling units.publishe