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Novel aqueous two-phase micellar systems (ATPMS) composed of Pluronic F68, a triblock amphiphilic copolymer, and cholinium-based ionic liquids (ILs) were formulated and applied for separation/purification of curcumin (CCM). CCM stability in the presence of ATPMS components was also evaluated. CCM is stable up to 24 h in copolymer (1.0 10.0 wt%) and ILs (0.1 3.0 M) aqueous solutions. Very mild phase separation conditions (close to room temperature) were achieved by adding cholinium ILs to the Pluronic F68 + McIlvaine buffer at pH 6.0 solution. The decrease of cloud-point temperature is dependent on the relative hydrophobicity of IL anion, [Hex] > [But] > [Prop] > [Ac] > Cl. ATPMS composed of more hydrophobic ILs ([Ch][Hex] > [Ch][But] > [Ch][Prop]) are most efficient in the partition of commercial CCM into polymeric micelles-rich phase. The best ATPMS (0.70 M [Ch][But] and 0.60 M [Ch][Hex]-based ATPMS) were then used to purify CCM from a crude extract of Curcuma longa L. Both systems were very selective to separate CCM from protein-based contaminants (selectivity values 25; purification yields 12-fold). Pluronic F68-based ATPMS are promising for selective separation of hydrophobic biomolecules by using cholinium-based ILs as adjuvants to adjust phase separation temperatures and biomolecules partition., This study was funded by the Coordination for Higher Level Graduate Improvements (CAPES/Brazil, finance code 001), National Council for Scientific and Technological Development (CNPq/Brazil) and the State of São Paulo Research Foundation (FAPESP/Brazil, processes #2014/16424-7, #2017/10789-1, #2018/10799-0, #2018/05111-9; #2019/05624-9, and #2019/08549-8). A.M. Lopes and J.F.B. Pereira are grateful for the language revision of native speaker H.S. Pacheco Neto., info:eu-repo/semantics/publishedVersion

Made available in DSpace on 2019-10-06T15:18:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-08 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Lipopolysaccharide endotoxins (LPS) are the most common contaminant pyrogenic compounds found in intracellular recombinant biomolecules purified from Gram-negative bacteria, such as Escherichia coli. Thus, the purification downstream processing should guarantee the effective removal of LPS from the final bioproduct, particularly, therapeutic biopharmaceuticals. Aqueous two-phase micellar systems (ATPMS) appear to be an excellent strategy to purify recombinant biopharmaceuticals from the cell lysate of E. coli, reducing high LPS concentrations. In order to demonstrate the effectiveness of ATPMS as a biopharmaceutical purification platform, the influence of inorganic salt electrolytes (NaCl, Li2SO4, KI, or KNO3) on the partitioning of green fluorescent protein (GFP) and LPS removal using ATPMS composed of n-decyl tetraethylene oxide (C10E4) was evaluated. The impact of different LPS concentrations on GFP partitioning was also studied. The addition of electrolytes (i.e., NaCl or Li2SO4) to the C10E4-based ATPMS have reduced the phase forming temperatures to very mild conditions (ca. 17.00 and 13.00 °C, for NaCl and Li2SO4, respectively). The selective partitioning ability of the proposed ATPMS was further demonstrated, where a complete removal of the LPS from the micelle-poor phase (REMLPS > 98%) and a preferential GFP recovery (RECGFP = 97%, KGFP > 7) to the micelle-poor phase was obtained. The GFP partitioning was even enhanced by increasing LPS loading (104–106 EU/mL), probably due to the formation of mixed micelles between LPS and C10E4. It is here demonstrated that a C10E4/buffer + salt-based ATPMS can be a useful and straightforward platform for the removal of endotoxin contaminants and the purification of recombinant biopharmaceuticals from E. coli cell lysates. Department of Biochemical and Pharmaceutical Technology School of Pharmaceutical Sciences University of São Paulo – FCF/USP Ronin Institute Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University – UNESP Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University – UNESP FAPESP: #2005/60159-7 FAPESP: #2007/51978-0 FAPESP: #2014/19793-3 CAPES: 0366/09-9