What Will It Take to Push Recombinant Endotoxin Testing Methods Forward

For regulators, the thinking around LAL alternatives is evolving.

Recently, the US Pharmacopeia (USP)announced a planned addition to the United States Pharmacopeia-National Formulary (USP-NF) covering the use of recombinant cascadereagent (rCR) and recombinant Factor C (rFC) as an alternative method to the limulus amebocyte lysate(LAL) test for detecting endotoxins. This means that it may become easier to use non-animal-based LAL tests, which use proteins from horseshoe crab blood to test for endotoxins in products that may be introduced to a patient's bloodstream.

Jordi Iglesias, Scientific Portfolio Specialist with Endosafe, warns that this should not be mistaken for total regulatory acceptance of recombinants to replace LAL testing.

"It is a method that is now described in the pharmacopeia," he said. "Companies will be more familiar with the method, but the validation requirements for products will stay the same."

For alternative methodslike recombinant testing, researchers must demonstrate that the alternative is as reliable as the traditional approach. Essentially, they need to prove their work. When using traditional methods, companies aren't required to submit as much information, since decades of data already support their reliability. Furthermore, many regions already accept traditional methods such as LAL, allowing a company to reuse the same data in reports to multiple agencies.

"The chapters on LAL in Europe, Japan, and the US are harmonized," he said. "They use the same words. This is not the case with recombinant. It will be compendial in the US and Europe, but there might be slight differences in the validation requirements. I don't want to say difficult, but it will generate more challenges for the users."

For now, some companies are willing to undertake the extra work needed to use recombinants to meet their sustainabilitygoals and adhere to the 3Rs animal use principles of Replace, Reduce, and Refine. Recombinants represent the Replacement aspect, as they don't require horseshoe crabs, which can help companies meet some of their 3Rs or sustainability goals.

The new Chapter <86> Bacterial Endotoxins Test Using Recombinant Reagents will not replace Chapter <85> Bacterial Endotoxinsbut will supplement that chapter with methods for using alternatives. However, this change only affects the USP-NF, which provides guidance to the US Food and Drug Administration (FDA) but is not itself a regulation. It is a positive first step, but many more are required before the FDA will waive the monograph requirement for recombinant testing.

From rabbit pyrogen tests to LAL to recombinants

The path to full acceptance of recombinant technology isn't new. About 40 years ago, a similar transition happened when LAL began gaining popularity over the previously accepted rabbit pyrogen test. That test, developed in the 1940s, required injecting a rabbit and monitoring it for fever for every single test. Although it was widely used, it has been largely replaced by LAL, which uses a small amount of horseshoe crab blood harvested from catch-and-release programs. Just this year, the European Pharmacopoeia (EP) removedthe rabbit pyrogen test from 57 texts in an effort to help phase it out entirely.

"I think (recombinant) will take less time to be validated than LAL, but the problem will be that USP and EP might have different compendial requirements for validation," Iglesias said. "The differences might be very slight, but it could be different. If you want to sell in the US and Europe, you will need to find a validation that covers all the requirements in these two markets."

The process has already begun in some areas. For example, clients using recombinants for water testing at Charles River benefit from the additional validation steps being built into the primary validation package, meaning no extra work is required for them.

The shift toward recombinant endotoxin testing is gaining momentum, but the path to full adoption requires careful validation and adherence to both existing and emerging guidelines. At Charles River, we've successfully navigated this journey by validating our Endosafe® Trillium™ recombinant cascade reagent (rCR), setting new standards for sustainability while maintaining rigorous quality control. You can learn more from our recent webinar with Jordi Iglesias, Achieving Compliance: The Step-By-Step Process to Validating rCR Endotoxin Testing, which provides practical, actionable insights into how we achieved this and how you can implement these methods in your facility.