Endotoxin-free E. coli Expression System

Arguably the most costly aspect of producing therapeutic oligonucleotides and recombinant proteins in E. coli is purifying the products from contaminating Lipopolysaccharide, LPS, also known as endotoxin. A component of the outer-cell membrane of the organism, LPS contaminates all recombinant products made in the organism. Humans are exquisitely sensitive to the lipid A portion of LPS that interacts with TLR4/MD-2 receptors to unleash a toxic immune response. In order to market pharmaceutical products, manufacturers must laboriously eliminate essentially all endotoxin from their products to meet established standards.

RCT has acquired rights to develop the FreE coli™ technology to engineer E. coli that are viable and robust in research and industrial culture conditions and yet devoid of the lipid A component of endotoxin. Due to a suppressor mutation the organism produces a substitute, non-toxic molecule to populate the outer membrane. Additional mutations prevent reversion to the lipid A-producing phenotype.

Data demonstrate that proteins, oligonucleotides, and outer membrane extracts of FreE coli™ technology hosts do not bind to TLR4/MD-2 receptors or stimulate human macrophages to produce TNFα. The purification of materials produced in FreE coli™ technology hosts is dramatically simplified. The FreE coli™ technology is proving beneficial for a number of applications, including use in cell-based screening, industrial scale production of proteins and oligonucleotides, and research scale production of protein and oligonucleotides.

For the most part, production strains using the FreE coli™ technology are identical to the wild type counterparts except that they lack endotoxic activity. These FreE coli™ strains exhibit normal growth rates, reach high ODs, and readily express a wide variety of proteins and oligonucleotides that typically express in E. coli.

RCT has developed the technology under broad patent rights covering the LPS Endotoxin-free bacterial strains acquired from the University of Michigan and development programs with FZB (Forshungzentrum Borstel). Specific commercial host strains have been created for out-licensing to industry. These are the subject of additional intellectual property protection.

RCT is seeking partners to apply and commercialize the FreE coli™ technology.

Inventors

Ron Woodard, Ph.D., University of Michigan
Uwe Mamat, Ph.D., Forshungzentrum Borstel

RCT Contact

Chad W. Souvignier, Managing Director