Protein solutions used for research, vaccines, or therapeutics need to be free of contaminants. One of the chief concerns is the presence of endotoxins (lipopolysaccharides) because their removal from protein solutions is a challenge. Typically, removal techniques utilize adsorption onto surfaces of beads in batch reactions, onto beads packed in columns, or onto membrane surfaces.
When poly(ε-lysine), currently used as a food preservative, is impregnated into cellulose beads, it can be safely used as a ligand for endotoxin removal using affinity chromatography. Poly(ε-lysine) contains the cationic binding sites necessary for endotoxin adsorption. Attaching the ligand to cellulose base beads achieves greater selectivity for endotoxins.
The biological activity of endotoxin is associated with the lipopolysaccharide (LPS), which is composed of a non-polar lipid (lipid A), a core polysaccharide, and heteropolysaccharide (O-antigen). The lipid A component imbues endotoxins with toxicity; immunogenicity is associated with the polysaccharide component.3 LPS is an amphipathic substance4 that possesses both anionic regions (the phosphoric acid groups) and hydrophobic regions (the lipophilic groups).
In physiological solutions, LPS aggregates form supramolecular assemblies (MW up to 1 x 106), with phosphate groups as the head group, and exhibit a negative net charge because of their phosphate groups. Aggregation of endotoxins is ascribed to the O-antigen end of the molecule, which gives it detergent-like abilities enabling micelle formation. Divalent cations have a role in stabilizing the aggregated structure of LPS while detergents destabilize the structure. Aggregation impacts not only the size of the endotoxins but also their chemical nature.
When E. coli and other gram-negative bacteria are used to produce recombinant proteins, it is particularly important to ensure that bacterial LPSs are removed from the final product. Until we adapted affinity chromatography to this task, no general method was available for the removal of endotoxins, particularly from protein solutions. Early techniques, usually adapted to specific products, can be found in the literature. These include ultrafiltration, solvent extraction, heat sterilization, solid phase adsorption, size exclusion, ion exchange, hydrophobic interaction, and reversed phase chromatography.