USP Permits Use of Non-Animal-Derived Reagents for Endotoxin Testing

The US Pharmacopeia (USP) announced on July 26, 2024 that its Microbiology Expert Committee has approved the inclusion of Chapter <86> Bacterial Endotoxins Test Using Recombinant Reagents into the United States Pharmacopeia–National Formulary (USP–NF). The chapter permits the use of non-animal-derived reagents for endotoxin testing; the finalized chapter will be published for early adoption in November 2024 before becoming official in May 2025. The new chapter is part of USP’s commitment to expanding the use of animal-free methods and materials.

Endotoxins may cause irreversible side effects and even death in severe cases at sufficient concentrations in the bloodstream (1). Bacterial endotoxins are found in the outer cell membrane of Gram-negative organisms and are known to be one of the most potent toxins. “These toxins are harmless outside of the body and in the alimentary canal. They are extremely potent, however, once introduced in the bloodstream or intrathecally,” Allen L. Burgenson, Testing Solutions, Lonza Pharma & Biotech, said (1).

“Bacterial endotoxins (also referred to as pyrogens) have the capacity to induce a high fever,” said John Dubczak, general manager, Microbial Solutions, Charles River Laboratories (1). “A large enough dose of endotoxin can lead to lung and kidney failure, intravascular coagulation, and a systemic inflammatory response that can lead to septic shock and death.”

Therefore, endotoxin testing is an important part of the quality and safety steps taken in the manufacture of certain sterile pharmaceutical products. Techniques for bacterial endotoxin testing using non-animal derived reagents are provided in the new chapter. Specifically, the new chapter discusses methods using both recombinant cascade (rCR) and recombinant Factor C (rFC) reagents. It also gives information on how to incorporate the methods into quality testing.These new techniques are in addition to those found in Chapter <85> Bacterial Endotoxins.

“Chapter <86>is intended to provide manufacturers with the information needed to use non-animal derived reagents. Manufacturers that currently use limulus amebocyte lysate (LAL) for endotoxin testing can continue to do so and Chapter <86> has no impact on them,” USP said in a press release (2).

One of the main endotoxin tests, LAL assay “works via the reaction of the LAL reagent-an aqueous extract of blood cells from the horseshoe crab-with bacterial endotoxins and lipopolysaccharides” (1). While the LAL test is an animal-derived test, and the animal itself is not tested upon, the desire to reduce animal use in testing has influenced the pharma industry, which resulted in the LAL test replacing the previous standard Rabbit Pyrogen Test. The LAL test is an animal-derived test, meaning the animal itself is not tested upon (1). This change prevented the use of hundreds of thousands of rabbits per year. There has also been a commitment to conserve the horseshoe crab. “Work has included sustainability partnerships to enhance the horseshoe crab population along the mid-Atlantic shores,” said Victoria Watson, laboratory manager, Microbiology at Wickham Labs. “Such projects have focused on increasing the survival rate of the horseshoe crab in early stages of life. Eggs are collected, and the crabs are grown in predation-free, controlled environments then released into their natural environment when older and stronger” (1).

References

1. Thomas, F. Putting Endotoxins to the Test. Pharm. Technol. 2019 43 (7). https://www.pharmtech.com/view/putting-endotoxins-test
2. USP. Expert Committee Approves Endotoxin Testing Using Non-animal Derived Reagents. Press Release. July 26, 2024.