Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 2. Nonionic micellar systems.

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]