LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

Abstract

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.

Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

“…TX-114 based phase separation was shown to be an efficient, simple and cost-effective strategy to significantly reduce or even eliminate endotoxin contamination from biological preparations [9, 16–18, 20–23, 26, 32]. Despite the scientific need for LPS removal to establish the immunological activity of natively folded proteins, only few studies reported on the experimental conditions that affect the efficiency of endotoxin removal from protein extracts using TX-114.…”

“…A previously published TX-114 based LPS extraction protocols [25, 26] was modified to optimize for LPS removal and removal of remaining TX-114. TX-114 (Amresco, cat.…”

Phage Therapy: Eco-Physiological Pharmacology

“…By contrast, there unquestionably can be toxicity associated with the bacterial lysis products that are produced in the course of phage-product manufacturing, potentially including bacterial exotoxins if those are generated by the bacteria used to produce phages. Also of concern is endotoxin that is solubilized upon lysis of Gram-negative bacteria [136, 140–142]. Generation of these lysis products in vivo , during treatment, is an issue associated with any lytic antibacterial and certainly should be taken into account during the design of phage therapy protocols and/or avoided via the use of lysis defective phages [139, 143].…”

Aqueous Two‐Phase Systems (ATPSs) for Extraction and Purification of Biomolecules and Bioparticles

“…Lopes et al (74) demonstrate the LPS removal efficiency of this system from a fermentation broth of recombinant E. coli producing GFP. This system was capable of, while extracting GFP at rates superior to 90%, removing more than 99,5% of LPS from the sample (75). Liu et al (76) studied the partition behavior of bacteriophages (such as X174, P22, and T4) on micellar ATPSs using n-decyl tetra (ethylene oxide) (C 10 E 4 ) as surfactant.…”