Alternative Endotoxin Testing Methods

Horseshoe crabs have played a role in human health since the 1970s.  LAL and TAL methods have changed since the first gel clot test, however the last advancement came in the 1980s when a chromogenic substrate was added to the LAL/TAL chemistry.  All of the LAL and TAL methods require the bleeding of animals.  Global conservation and preservation of the horseshoe crab can be attained with alternative methods that can reduce or eliminate the need to use an animal for endotoxin testing.

There are alternative and sustainable methods available for those pharmaceutical, medical device, and dialysis companies willing to embrace sustainable, animal-friendly innovations in endotoxin detection.

Recombinant Factor C (rFC) Methods Derived from the Horseshoe Crab
The National University of Singapore developed and Lonza commercialized an endotoxin test that does not require horseshoe crab blood.  Instead, the DNA for one of the horseshoe crab blood clotting factors, Factor C, was cloned and is manufactured recombinantly (synthetically).  Recombinant Factor C (rFC), similar to the native Factor C, is activated by endotoxin. In the rFC test method, branded PyroGene™ by Lonza, activated rFC cleaves a fluorogenic substrate and the fluorescent signal is monitored and analyzed to quantitate endotoxin content.  Recombinant technology is used to manufacture therapeutics.  It makes sense to use similar technology to help assess the safety of products.  The horseshoe crab provides some DNA to support this alternative advancement in endotoxin detection.  Widespread adoption of this method could help significantly reduce the need to bleed horseshoe crabs.  In areas of the world where horseshoe crabs are limited and/or declining, switching to the rFC method could help make a positive impact to the crab population.  For more information, visit www.lonza.com/hsc.

Another company developing and supplying endotoxin detection assays based on rFC is Hyglos (www.hyglos.com). In addition to the rFC assay EndoZyme®, Hyglos applies endotoxin-binding molecules derived from bacteriophagesfor specific binding to the endotoxin present in the sample, with EndoLISA®. By removing the interfering sample matrix prior to detection, the EndoLISA® assay has revolutionized endotoxin testing of complex samples that typically inhibit detection when using conventional methods,. The EndoLISA® methodology for removing interfering factors is included in the European Pharmacopoeia chapter 5.1.10., section 9.

The methods based on rFC have recently been included in the European Pharmacopoeia guidelines as valid alternatives to the LAL test. In order to be used for product release, the rFC methods must be validated and show to provide equivalent or better performance than LAL.

Monocyte Activation Test
Another method that does not require the use of horseshoe crab blood is the Monocyte Activation Test (MAT) or the In Vitro Pyrogen test.  This test method uses human blood rather than horseshoe crab blood.  The MAT method measures the release of cytokines from blood cells due to the presence of pyrogens, such as endotoxin, in the test sample.  The MAT is basically mimicking what occurs in our blood stream when it is exposed to pyrogenic substances.  The MAT has an advantage over the LAL/TAL and rFC methods as it can detect non-endotoxin pyrogens. Currently, there are no non-endotoxin pyrogen standards and to date the Monocyte Activation Test has not been able to show a reaction with an authentic non-endotoxin pyrogen.

Industry Reluctance to use Alternative Testing Methodologies is Shifting

Adopting an alternative endotoxin detection method does have regulatory implications for companies willing to move away from traditional LAL/TAL methods. The LAL/TAL methods are included in the harmonized chapters of pharmacopoeia globally including the United States, Europe and Japan. Regulators, such as the U.S. Food and Drug Administration, follow the test chapters in the pharmacopoeia publications. Changing to an alternative method requires validation (additional testing to show equivalence to the compendial method). Biomedical/pharmaceutical companies appear increasingly willing to devote time and resources to qualify an alternative method as the regulatory acceptance is growing. The pharmacopoeias include chapters that directly address what is needed to qualify an alternative method so that it will be accepted by regulators.

Whether qualifying and adopting an alternative method for endotoxin testing on final products or using an alternative method for raw material or in process testing, increasing the use of alternative methods can help reduce the demand on horseshoe crab blood.

Eli Lilly and Company Validation/ Equipment Strategy Presentation

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See our list of companies that purchase their endotoxin detection materials from sustainable and responsible manufacturers.

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