Bacterial endotoxin test (revision 30)

The bacterial endotoxin test detects lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria that provokes a febrile response when introduced parenterally. The test exploits a clotting cascade in amoebocyte lysate derived from horseshoe crab blood, which is activated by picogram quantities of endotoxin.^[1]

Endotoxin is not a living organism and is not removed by sterilisation. A preparation can be sterile and pyrogenic at the same time: autoclaving kills bacteria but leaves their lipopolysaccharide intact and biologically active. Endotoxin is the commonest but not the only pyrogen, and a separate rabbit pyrogen test exists for the wider class.^[2] This is why endotoxin and sterility are separate determinations answering separate questions.^[1]

Results are expressed in endotoxin units, normalised to mass for a drug substance or to volume for a solution. Compendial limits for parenteral products are calculated from the maximum dose, and a research-chemical certificate reporting an endotoxin figure without the basis of normalisation is difficult to interpret.^[3]

Three variants are recognised. The gel-clot method observes whether a firm clot forms at a given dilution and gives a limit result rather than a number. The turbidimetric method follows the increase in turbidity as the cascade proceeds and is quantitative. The chromogenic method measures release of a coloured product from a synthetic substrate and is also quantitative.^[1]

Recombinant factor C assays, which use a recombinant version of the initiating enzyme rather than crab-derived lysate, have been introduced and are increasingly accepted. They avoid the animal-derived reagent and are more specific, since they respond only to endotoxin and not to β-glucans.

Interference is the routine difficulty. Sample matrices can enhance or inhibit the cascade, so a validated method includes a positive product control demonstrating recovery of a known spike. A result reported without evidence that interference was assessed is not fully interpretable.^[1]

Endotoxin contamination arises from the water and the equipment, not usually from the synthesis. Water is the principal route: bacteria grow readily in stagnant water systems, and even after they are killed their lipopolysaccharide persists. Glassware carries endotoxin unless depyrogenated by dry heat, which is a more aggressive process than sterilisation.^[1] Water-system control is accordingly treated as a manufacturing rather than a testing problem.^[4]

Lyophilised research peptides are not manufactured as parenteral products and are not required to meet a parenteral endotoxin limit. Some certificates report endotoxin nonetheless; many do not. Absence of the determination is not evidence of a problem, and its presence is not evidence that the material is suitable for any particular purpose.

For comparison, the compendial limit for most parenteral products corresponds to 5 EU per kilogram of body weight per hour, translated into a per-milligram limit by the maximum dose. A figure quoted in EU/mg without the dose basis cannot be compared against that limit by a reader.^[3]

A low endotoxin figure establishes that the sample tested contained little detectable lipopolysaccharide. It does not establish sterility — see Sterility testing — and does not address non-endotoxin pyrogens, which the test does not detect at all.^[1]

Nor does it transfer between vials. Endotoxin contamination is frequently heterogeneous, arising from a particular piece of equipment or a particular fill, so a determination on one unit constrains other units only through the assumption of lot uniformity that the whole apparatus of lot traceability exists to support.

The general caution about detection limits applies here too: "not detected" means below the sensitivity of the method used, and the method sensitivity is a field the report should state. See Limit of detection.^[3]

• USP General Chapter 85
• Sterility testing
• USP General Chapter 71
• Limit of detection
• Certificate of analysis