LPS removal from an E. coli fermentation broth using aqueous two-phase micellar system.
In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two-phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (K(GFPuv) < 1.00), and LPS removal into the micelle-rich phase (%REM(LPS) > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.
Lopes
André M
AM
Dept. of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, FCF/USP, São Paulo/SP, Brasil. andreml@usp.br
Magalhães
Pérola O
PO
Mazzola
Priscila G
PG
Rangel-Yagui
Carlota O
CO
de Carvalho
João C M
JC
Penna
Thereza C V
TC
Pessoa
Adalberto
A
D001709:Biotechnology; D005591:Chemical Fractionation / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D005285:Fermentation; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008823:Micelles; D014867:Water / Q000737:chemistry
D008070:Lipopolysaccharides; D008823:Micelles; D014867:Water; D049452:Green Fluorescent Proteins