Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by non flammable solvents.

Abstract

During the production of recombinant proteins from gram negative bacteria, lipopolysaccharides (LPS, endotoxin) are released along with the protein of interest. In many instances, LPS will copurify with the target protein due to specific or non-specific protein-ILPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on anion or cation exchange chromatographic media. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile due to their lower toxicity and decreased flammability. In addition, they are less costly than many of the detergents that have been used for such purposes. LPS removal efficiency increased with increasing alkane chain length. 1,2-alkanediols were more effective than terminal alkanediols in the separation of LPS from protein LPS complexes.

Description

FIELD OF THE INVENTION [0001] Embodiments of the present invention generally relate to the removal of lipopolysaccharides (LPS) from protein-lipopolysaccharide complexes. BACKGROUND OF THE INVENTION [0002] Lipopolysaccharide (LPS) is a major component of the outer membrane of gram negative bacteria. The endotoxic component of LPS is the lipid A portion. It comprises 1,6-linked D-glucosamine residues that are substituted with up to six acyl chains and a core polysaccharide structure to which additional polysaccharide repeating units may be attached. Endotoxin is a potent activator of the innate immune system at low doses while at higher doses endotoxin induces a number of other physical reactions including septic shock and death (Heine et al., 2001). Contamination of therapeutic products with endotoxins is therefore a primary concern for the manufacturers of such products. [0003] Many recombinant proteins are produced in the gram negative bacteria Escherichia coli. The removal of LPS...

AI Technical Summary

Benefits of technology

[0007] Embodiments of the present invention generally relate to the ability of alkanediols to separate lipopolysaccharides (LPS) or other endotoxins from proteins. In an embodiment, alkanediols are able to effect the separation of LPS from LPS-protein compl

Problems solved by technology

The removal of LPS from these recombinant proteins can be a complicated but essential process especially if the proteins are destined for therapeutic uses.

Often, during the production of recombinant proteins, difficulties in the separation of LPS from proteins are encountered due to protein-LPS interactions.

Some experiments have shown that alcohol and detergent washes during ion exchange chromatography are effective in reducing the protein associated LPS levels while poor separation of LPS from the proteins was obtained by the denaturing HIC procedure.

Even though the alcohol and detergent washes were successful at reducing the levels of LPS in the LPS-protein complexes, scaling up and implementing any of these procedures in a manufacturing setting would not be practical.

At these concentrations, these solutions are considered flammable liquids and as such impose many safety and operational restrictions.

The detergents, even though very effective at reducing LPS levels, are relatively expensive and would add significant cost to a manufacturing process and may affect the bioactivity of the protein of interest.

Images