Affinity Chormatography Removes Endotoxins

Porous cellulose beads impregnated with poly(?-lysine) are a selective packing to purify protein solutions.

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.

Figure 1. Schematics of (a) Gram-Negative Bacterial Cell Wall and (b) Lipopolysaccharide1
ENDOTOXINS Pyrogens are a class of substances that can raise body temperature. They commonly originate from constituents of gram-negative cell walls but can also leach from some chemicals and materials. Figure 1 illustrates a gram-negative bacterial cell wall and a detail of a lipopolysaccharide.1 Gram-negative bacteria shed portions of their cell walls and lipopolysaccarides into their environment, flooding it with endotoxins. Endotoxins are potent stimulants of mammalian immune systems, causing pyrogenic and shock reactions. Their removal in parenterals is mandated by FDA, which has established critical limits of pyrogen load for parenteral products.2

Pyrogens in parenterals and medical device products can come from a variety of sources. They often are contributed by the raw materials, process water, excipients, cell culture and fermentation additives, chromatography media, process equipment, and packaging components. Rigorous control of microbiological contamination will effectively minimize levels of endotoxin contamination.

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).

Table 1. Endotoxin Limits for Various Products6
A small amount of endotoxin, about 0.1 ng per kg of body weight, can cause a pyrogenic reaction. The standard reporting unit for endotoxin data is one endotoxin unit (EU), the equivalent of 0.1 ng. A typical gram-negative bacterium contains 10-15 g of LPS, which means that at least 105 bacterial cells are required to contribute 0.1 ng.5 This is the basis of the endotoxin level requirements listed in Table 1.

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.

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