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Graphical abstract Highlights • GFP recovery and LPS removal on C 10 E 4 /buffer + salts-based ATPMS were studied. • C 10 E 4 /buffer + salts promoted GFP partitioning to the micelle-poor phase (K GFP > 7). • C 10 E 4 /buffer + salts-based ATPMS removed LPS to the micelle-rich phase (REM LPS > 98%). • High loads of LPS (>106 EU/mL) increased the GFP partition into micelle-rich phase. • C 10 E 4 /buffer + salt are able to separate selectively the GFP from contaminant LPS. Abstract 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, Li 2 SO 4 , KI, or KNO 3) on the partitioning of green fluorescent protein (GFP) and LPS removal using ATPMS composed of n -decyl tetraethylene oxide (C 10 E 4) was evaluated. The impact of different LPS concentrations on GFP partitioning was also studied. The addition of electrolytes (i.e., NaCl or Li 2 SO 4) to the C 10 E 4 -based ATPMS have reduced the phase forming temperatures to very mild conditions (ca. 17.00 and 13.00 °C, for NaCl and Li 2 SO 4 , respectively). The selective partitioning ability of the proposed ATPMS was further demonstrated, where a complete removal of the LPS from the micelle-poor phase (REM LPS > 98%) and a preferential GFP recovery (REC GFP = 97%, K GFP > 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 C 10 E 4. It is here demonstrated that a C 10 E 4 /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. [ABSTRACT FROM AUTHOR]

Aqueous two-phase micellar systems (ATPMS) are more than simple amphiphilic copolymer or surfactant aqueous solutions since due to the formation of biphasic regimes, at certain temperatures, these can be successfully used for the separation, encapsulation, and formulation of several bio-based compounds. The recent use of ionic liquids (ILs) as adjuvants in the formation of ATPMS have enlarged its "working window", but the measurement of respective binodal curves is still very empiric and dependent of operator accuracy through the visual determination of cloud-point temperature. In this work, the range of ATPMS using ILs as adjuvants was extended, providing more insights about their phase formation mechanisms, as well as into determination of cloud-point values of the lower critical solution temperature (LCST)-type demixing transition by using the new temperature modulated optical refractometry approach. The binodal curves of ATPMS composed of Pluronic® L35/PBS buffer at pH 7.4 + cholinium ILs were determined, by using two different methods, i.e.: cloud-point temperature (T CP) visual determination versus temperature modulated optical refractometry (TMOR). TMOR method was more sensitive to the determination of biphasic transition, particularly, in highly diluted solutions (< 3.0 wt% of Pluronic L35). The results also established that the addition of cholinium ILs decreased significantly the phase separation temperatures of Pluronic L35/PBS, with an increase of biphasic region (viz. lower of T CP values) as a function of the relative hydrophobicity of the IL anion, i.e. : [Hex]− > [But]− > [Pro]− > [Ac]− > Cl−. The use of cholinium ILs as adjuvants is particularly important to provide mild conditions for ATPMS-based separation and encapsulation strategies, while TMOR-based methods allow an improvement of phase separation determination accuracy. Unlabelled Image • Refractometry index Vs. cloud-point temperature by visual technique were compared. • The refractometry-based technique is more accurate to determine the binodal curves. • Cholinium ILs decreased the phase separation temperature of Pluronic L35/PBS. • Cloud-point temperature decreased with the increase of the IL anion hydrophobicity. [ABSTRACT FROM AUTHOR]

• Partition of curcumin (CCM) using ATPMS composed of Pluronic F68 + ILs was studied. • Cholinium ILs decreased the phase separation temperature of Pluronic F68/buffer. • The phase separation decreased with the increase of the IL anion hydrophobicity. • CCM was preferentially partitioned into polymeric micelles (PMs)-rich phase. • ILs-based ATPMS were very selective to recover CCM from crude extract of C. longa L. 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]− > [Pro]− > [Ac]− > Cl−. ATPMS composed of more hydrophobic ILs ([Ch][Hex] > [Ch][But] > [Ch][Pro]) are most efficient in the partition of commercial CCM into polymeric micelles (PMs)-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. [ABSTRACT FROM AUTHOR]