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Endotoxin testing

Endotoxin testing is the measurement of bacterial endotoxin in a sample, most often with a Limulus amebocyte lysate (LAL) assay. Endotoxin is the lipid A domain of lipopolysaccharide, a molecule of the outer membrane of Gram-negative bacteria.[1],[2]

What is endotoxin?

A review by Raetz and Whitfield describes bacterial lipopolysaccharides as typically consisting of a hydrophobic domain known as lipid A, or endotoxin, a nonrepeating core oligosaccharide and a distal polysaccharide, the O-antigen. The review also identifies the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells.[2]

How does an LAL assay detect it?

Levin and Bang reported in 1964 on the role of endotoxin in the extracellular coagulation of the blood of Limulus, the horseshoe crab.[3]

Young, Levin and Prendergast reported in 1972 that lysates of Limulus amebocytes are gelled by endotoxin, and that the data suggest endotoxin activates an enzyme which then gels a clottable protein in the lysate. The rate of the reaction depended on the concentration of endotoxin, and the reaction was temperature and pH dependent.[4]

Which methods and standards does FDA name?

FDA guidance states that for more than 30 years FDA has accepted an LAL test for endotoxins in lieu of the rabbit pyrogen test. It names USP chapter <85>, Bacterial Endotoxins Test, and describes gel clot, photometric and kinetic test methods. The rabbit test is USP chapter <151>, Pyrogen Test.[1],[5],[6]

The guidance states that when results differ between methods, or in the event of a dispute, the final decision is made on the USP compendial gel-clot method unless the monograph for the product says otherwise. It also states that manufacturers may use another endotoxin test after demonstrating a reproducible correlation between methods and the USP reference standard.[1]

ICH Q6B names endotoxin detection as an example of a pharmacopoeial test that should be performed on a drug substance where appropriate.[7]

What can interfere with a result?

The same FDA guidance states that bacterial endotoxins assays are subject to a variety of interferences related to the physical and chemical properties of the test article, and describes inhibition and enhancement testing.[1]

Bolden and colleagues describe spike and hold studies in which a known amount of purified lipopolysaccharide is added to a sample to show that endotoxin can be recovered by the LAL method over time, and report that sample preparation details such as mixing time affected the result.[8]

How is a result reported?

Endotoxin limits in the FDA guidance and in USP chapter <161> are expressed in endotoxin units, for example EU/mL.[1]

The PepGenex certificate reports endotoxin by LAL against a limit of 5.0 EU/mL.

Limitations

An endotoxin test measures endotoxin, the lipid A of Gram-negative bacterial lipopolysaccharide. ICH Q6B lists endotoxin and microbial limits as separate typical pharmacopoeial tests; an endotoxin result is not a microbial result.[2],[7]

A result applies to the sample and dilution tested, within the method's validated range and after interference testing.[1],[8]

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Research peptide profiles in this library. What this page describes applies to peptide lots in general; none of these profiles reports a result of it for any lot.

References

  1. U.S. FDA. Pyrogen and Endotoxins Testing: Questions and Answers. Guidance for industry. FDA docket FDA-2013-S-0610
  2. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635-700. PMID 12045108 · DOI 10.1146/annurev.biochem.71.110601.135414 · PMC2569852
  3. Levin J, Bang FB. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull Johns Hopkins Hosp. 1964;115:265-274. PMID 14209047
  4. Young NS, Levin J, Prendergast RA. An invertebrate coagulation system activated by endotoxin: evidence for enzymatic mediation. J Clin Invest. 1972;51(7):1790-1797. PMID 4624351 · DOI 10.1172/JCI106980 · PMC292326
  5. United States Pharmacopeia, General Chapter <85> Bacterial Endotoxins Test (as named in FDA, Pyrogen and Endotoxins Testing: Questions and Answers). USP <85>
  6. United States Pharmacopeia, General Chapter <151> Pyrogen Test (as named in FDA, Pyrogen and Endotoxins Testing: Questions and Answers). USP <151>
  7. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. U.S. FDA guidance for industry, August 1999. FDA docket FDA-1998-D-0003
  8. Bolden JS, Warburton RE, Phelan R, et al. Endotoxin recovery using limulus amebocyte lysate (LAL) assay. Biologicals. 2016;44(5):434-440. PMID 27470947 · DOI 10.1016/j.biologicals.2016.04.009