Endotoxin Testing (LAL)

Endotoxin testing (LAL test) ensures injectable therapeutics are safe for human use.

Bacterial endotoxins are lipopolysaccharides (LPS), components of Gram-negative bacterial cell walls known to cause fevers and disease when injected into the bloodstream. Bacterial endotoxins are heat stable and toxicity is not dependent on the presence of the bacterial cell.

Since many therapeutics are made in bacteria, endotoxin testing is employed to ensure a therapeutic product is endotoxin-free. BioReliance tests for endotoxin in several areas of drug manufacturing and production including:

  • Bulk Lot Release Testing
  • Final Product Release Testing
  • Raw Materials Testing

BioReliance research professionals have performed thousands of endotoxin testing assays for clients in Biopharma. We can provide a fast turnaround time and the highest quality testing to produce therapeutics that are bacterial endotoxin-free and safe for use in humans.

To see our endotoxin testing assays, use the search tool above.

How is endotoxin testing performed (the LAL test)?

The most common approach to endotoxin testing is the limulous amoebocyte lysate test (LAL test). This assay is based in the biology of the horseshoe crab (Limulous). These animals produce LAL enzymes in blood cells (amoebocytes) to bind and inactivate endotoxin from invading bacteria.

LAL serves as a primitive immune system. Inactivation of endotoxin also forms a clot, which can further protect the horseshoe crab from infection. The LAL test exploits the action of this enzyme, by adding LAL reagent to the tested product, and assaying for clot formation.

BioReliance offers LAL - endotoxin tests with a variety of assay options including:

  • Quantitative and qualitative testing
  • Gel-clot method (LAL test)
  • Chromogenic methods (USP Chromogenic)

The LAL test was accepted by the FDA for endotoxin testing of pharmaceuticals and medical devices in 1983. USP requires pooled testing of a production lot for the presence of bacterial endotoxin.

To find the right endotoxin testing assay, use the search tool to the right or click here.

Endotoxin testing - the gel clot method vs. the chromogenic assay.

Both the gel clot method and the chromogenic method of endotoxin testing are appropriate for preclinical, Phase 1, Phase 2, Phase 3, and Licensed Product phases of therapeutic production and distribution.

Either test is approved for endotoxin testing of multiple therapeutic types including cell therapy, recombinant protein, monoclonal antibody, vaccines and gene therapy. The choice comes down to whether you need quantitative results or simply to detect endotoxin in your drug.

The gel clot assay with the LAL test is simplest method for the detection of bacterial endotoxins and the GMP format is typically used for lot release of final product intended for injection in humans.

To perform the assay, equal volumes of LAL reagents are mixed with undiluted or diluted test article and observed for clot formation. The dilutions are selected to cover the potential range of endotoxin in the sample and to reduce interference by the test material making the gel clot LAL test semi-quantitative. The levels of endotoxin allowed are strictly regulated and depend on the route and frequency of administration. The sensitivity of this assay is 0.06 EU/ml.

The USP chromogenic method is based on the activation of a serine protease (coagulase) by the endotoxin, which is the rate-limiting step of the clotting cascade. The assay measures the activation of the serine protease as opposed to the end result of this activation, which is clotting. The natural substrate, coagulogen, is replaced by a chromogenic substrate. On cleavage of this substrate a chromophore is released from the chromogenic peptide and is measured by spectrophotometry.

The USP chromogenic method is quantitative and can provide a greater sensitivity over a wider range. The sensitivity of this assay is 0.10 EU/ml.

BioReliance CRO is an expert in both types of endotoxin tests and can provide you with advice on the best assay for your drug study or lot release needs.

Request information about endotoxin testing by using the search tool above or click here to contact us.

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