The Evolution of Eco-Conscious Endotoxin Testing

Over the past century, few species have experienced such a profound shift in public perception as the American horseshoe crab (HSC). Before 1920, millions of Limulus polyphemus were harvested for livestock feed and fertilizer.¹ By the late 1940s, bounties were paid for their destruction due to their perceived threat to commercially important species like softshell clams.² Today, while fisheries continue to harvest adult horseshoe crabs for bait, public interest in conserving this unique species has grown significantly.

Establishment of Conservation Efforts

The Atlantic States Marine Fisheries Association (ASMFC) began management of the species in 1998. In 2011, LAL manufacturers collaborated with ASMFC to establish “Best Management Practices for Handling Horseshoe Crabs for Biomedical Purposes.”

Over time, these best practices became embedded in the Standard Operating Procedures for LAL manufacturers. In 2023, a new group of LAL manufacturer representatives, fisheries biologists, and natural resource managers revisited the document. Although the 2023 meetings did not produce significant practice changes - reflecting the foresight of the 2011 group - the participants updated the document to accurately reflect current LAL collection practices and serve as a public-facing educational resource.⁴

As part of ASMFC’s management, regular stock assessment reports assess the population status and pressures like commercial fisheries. In the 2019 stock assessment, statistical modeling using the Adaptive Resource Model (ARM) determined that the biomedical collection of horseshoe crabs had no measurable impact on the population.¹ The SMEs have also indicated that all estimated mortality from LAL manufacturing along the US East Coast applied solely to this sub-population would represent about 0.3 percent of the adult population.⁵ These findings were further supported by ASMFC’s 2024 stock assessment update.⁶

The Evolution of Eras

Despite findings that LAL manufacturing has a negligible effect on HSC populations, there is still a need to move towards alternatives that do not use animal-derived products. For example, early LAL tests, especially gel-clot assays, utilize large amounts of crude LAL, limiting the number of tests produced per liter of raw lysate. However, subsequent innovations introduced kinetic chromogenic assays with a lower ratio of crude lysate required for each test. Chromogenic tests represent a 54% reduction in lysate used per assay produced compared to turbidimetric and gel-clot assays. Additionally, microfluidic assays have been available for over 20 years and represent a 95% reduction in raw lysate used per test compared to traditional counterparts. With improved formulations and various commercially available recombinants, options requiring no crude lysate are now available.

An Introduction to Recombinant Testing

The use of recombinant bacterial endotoxin testing (BET) has come a long way for the pharmaceutical and biotech industries. Historically, the LAL assay has dominated testing for endotoxins in parenteral drugs, medical devices, and other biologicals. However, Recombinant Factor C (rFC) methods introduced in the early 2000s and Recombinant Cascade Reagent (rCR) methods introduced in the late 2010s have gained traction as alternative methods. Recombinant BET relies on genetically engineered enzymes that mimic the endotoxin detection properties of LAL. The journey to widespread adoption of recombinant endotoxin tests has been gradual and filled with regulatory and technical challenges; however, strides are being made to make it easier for end users to transition over.

Regulatory and Testing Development

In the early 2000s, recombinant methods faced limited availability, with only one primary vendor offering these products. Although rFC has been available since 2003, widespread adoption was hindered by regulatory hurdles, limited options, and the need for significant investments in equipment, training, and validation protocols. The transition from the LAL method to rFC is not as simple as switching from one reagent to another. Moving to an rFC requires a significant investment in new equipment, processes, training efforts, and validation protocols. The introduction of rCR methods in the late 2010s provided a path for maintaining existing equipment and processes, but the need for alternative method validation remained a challenge. Manufacturers must demonstrate that recombinant methods meet the same standards for sensitivity, precision, reproducibility, and equivalency established over decades with LAL.

LAL has long been the gold standard for BET due to its compendial status, decades’ worth of data, and no reported cases of failure due to false negative tests. The United States Pharmacopeia (USP), European Pharmacopeia (EP), Japanese Pharmacopeia (JP), and other global standards recognized LAL as the primary endotoxin detection method in biologics, whereas recombinant methods were not compendial and lacked formal recognition. Because recombinant methods were not compendial, end users choosing to utilize these methods had to navigate the complex process of a full alternative method validation and regulatory submission following the guidance of local regulators. While regulatory agencies have provided some guidance on method validation, there is a lack of clear understanding and standardized framework available detailing the switch from LAL to recombinant methods. Hesitation among many drug manufacturers made it difficult to justify the move to recombinant methods and risk investing in new technology with minimal regulatory acceptance, only to encounter roadblocks during review or face potential rejection altogether.

A Priority Paradigm Shift

As recombinant technologies gained recognition as a more sustainable method for BET, the situation began to shift on a global scale. Pressure from investors and regulators is pushing companies to adopt environmental, social, and governance strategies that call for the reduction or replacement of animal testing. Now, with multiple vendors offering recombinant solutions, end users gain more options and flexibility. This expansion in the vendor landscape allowed end users to test their products against several recombinant options on the market and select the best fit for their specific needs. The ability to source from different suppliers gave companies a level of supply chain security that had been missing when only one vendor or recombinant option was available.

Recombinant BET is becoming more widely used and accepted by the endotoxin testing industries as end users begin comparative studies and adoption for routine testing, and regulatory guidelines continue to evolve. Companies’ requirements for more sustainable BET methods will also continue to drive the move from LAL to recombinant technologies. It is important to note that that transition will likely be slow as companies weigh the benefits of implementing new technology in their laboratories. Thus, LAL will continue to be available for the foreseeable future, given its critical role in endotoxin testing and aid in legacy testing, validation, and comparative studies between methods.

The evolution of BET has been complex, driven by a variety of technical, regulatory, and sustainability obstacles; however, as the technology continues to mature and regulatory clarity and acceptance increase, recombinant methods will become a more common and trusted tool in ensuring safety and quality of biologic and pharmaceutical products.

Current Regulatory Status

Before the release of the USP Chapter <86> in late 2024, EP Chapter 2.6.32 was released as a compendial chapter in Europe in 2021 but currently only includes rFC. This poses a problem for companies that sell in both Europe and North America, and even with compendial chapters being adopted, the global regulations are far from harmonization. Another hurdle to implementation is that these chapters were not included in monographs at the time of their release. EP Chapter 2.6.32 was added to the “Water for Injection” (WFI) monograph just recently in 2024, but that only affects end users submitting products for approval in Europe. The approval of the USP Chapter <86> Bacterial Endotoxins Testing Using Recombinant Reagents, which includes both rFC and rCR, as a compendial chapter in July 2024 is a great step toward the further adoption of recombinant reagents. USP Chapter <86> guides the requirements, and it explicitly calls out that recombinant is still considered an alternative test method unless specified in an individual monograph. Additionally, it must meet the requirements outlined in USP General Notices 6.30. It states that the user of recombinant reagents must first review the recombinant supplier’s primary validation package to confirm that the reagent will be appropriate for use in testing their specific product or material. The recombinant supplier’s primary validation package should include the parameters highlighted in ICH Q2: Validation of Analytical Procedures and USP Chapter <1225>: Validation of Compendial Methods,s including accuracy, precision, equivalency, and robustness. Each recombinant reagent and supplier is different, so end users should thoroughly review and perform a risk assessment on any supplier primary validation package that they are using to supplement their test to ensure it meets all internal and external criteria. Second, the chapter references <1226> Verification of Compendial Procedures as a guide on how to perform a verification that the method is appropriate for use to test a specific product or material, commonly known as method suitability. More detailed information can be found in the FAQs for the USP Chapter <86>, but it is imperative to discuss submission strategies with the respective regulatory agency to ensure the determined path covers all requirements necessary for product submission. 

Although Recombinant Factor C has been around since 2003, the industry has not seen a significant shift towards using recombinant reagents until recently. To continue pushing for the inclusion of USP Chapter <86>  into monographs as quickly as possible, the USP welcomes all data generated from vendors and manufacturers for recombinant testing on various sample types. This input will further fuel the case for including USP <86> Chapter in individual monographs, thereby making recombinant technologies fully compendial and easier to adopt.

Conclusion

The development of sustainable practices in resource conservation and scientific innovation has allowed bacterial endotoxin testing to evolve significantly since its birth. Testing for endotoxins in the pharmaceutical market has been dominated by LAL; however, recent advances in technology and the addition of recombinant methodology to the market have proven positive for the industry. Regulations surrounding this testing are being updated to include recombinant technology on a global scale, with the most recent update being the USP addition of Chapter <86>, which provides the industry with critical information on the successful implementation of these technologies.

References

1. Atlantic States Marine Fisheries Commission. 2019. Horseshoe Crab Benchmark Stock Assessment and Peer Review Report. Arlington, VA.
2. Botton, M., Shuster, C. N., & Keinath, J. 2004. The American Horseshoe Crab: Horseshoe Crabs in a Food Web: Who Eats Whom? In C. N. Shuster, H. J. Brockmann, & R. B. Barlow (Eds.), The American Horseshoe Crab. Hardcover. 1st ed. Harvard University Press. 2004:133-151.
3. Atlantic States Marine Fisheries Commission. 2011. Horseshoe Crab Biomedical Ad-Hoc Working Group Report. Washington, DC.
4. Atlantic States Marine Fisheries Commission. 2023. Best Management Practices for Handling Horseshoe Crabs for Biomedical Purposes. Arlington, VA.
5. Atlantic States Marine Fisheries Commission. 2021. Meeting Minutes: ASMFC Horseshoe Crab Board Meeting, October 2021. Available at: https://www.asmfc.org/uploads/file/63d 2e49eHorseshoeCrabBoardProceedingsOct2021.pdf (p. 9).
6. Atlantic States Marine Fisheries Commission. 2024. 2024 Horseshoe Crab Stock Assessment Update. Arlington, VA.

Author Details 

Courtney Wachtel- Product Manager, Endosafe, Microbial Solutions, Charles River

Publication Details

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