Recombinant bacterial endotoxin testing (BET) has undergone significant evolution in the pharmaceutical and biotech industries, where quality control and safety are paramount.
Historically, testing for endotoxins in parenteral drugs, medical devices, and other biologicals has been dominated by the Limulus Amebocyte Lysate (LAL) assay derived from the blood of horseshoe crabs.
Recombinant bacterial endotoxin testing (BET) has undergone significant evolution
While LAL remains the gold standard due to its compendial status and decades of data, Recombinant Factor C (rFC) methods, introduced in 2003, and Recombinant Cascade Reagent (rCR) methods, introduced in the late 2010s, offer sustainable alternatives. 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, recent progress is focusing on easing the transition to recombinant methods.
Understanding current regulatory standards: USP, EP, and beyond
The regulatory landscape for recombinant BET has advanced with the introduction of compendial chapters like European Pharmacopeia (EP) Chapter 2.6.32 in 2021 and United States Pharmacopeia (USP) Chapter <86> in 2024.
These chapters describe the use of rFC and rCR, though challenges still remain, including the lack of harmonisation between global regulations and delays in incorporating recombinant methods into monographs. For example, EP Chapter 2.6.32 was only recently included in the Water for Injection (WFI) monograph in 2024, limiting its application to Europe.
General Notices provide the basic assumptions and definitions for applying USP-NF compendial standards, which are applied to official articles recognised in monographs and any applicable General Chapters.
General Notices provide the basic assumptions and definitions for applying USP-NF compendial standards
USP General Notices 6.30 states that users of recombinant reagents must review the primary validation package provided by the supplier to confirm the reagent’s suitability for testing specific products or materials. These validation packages should align with the parameters outlined in ICH Q2: Validation of Analytical Procedures and USP Chapter <1225>: Validation of Compendial Methods, covering aspects such as accuracy, precision, equivalency, and robustness.
Since recombinant reagents and suppliers vary, users are advised to perform a detailed risk assessment and thorough review of supplier validation packages to ensure compliance with both internal and external standards. Additionally, USP Chapter <86> references <1226>: Verification of Compendial Procedures for guidance on verifying method suitability for specific products or materials. The FAQs for USP Chapter <86> are a valuable resource for further details. It is also crucial to consult with regulatory agencies to ensure submission strategies align with all required criteria for product approval.
To accelerate the integration of USP Chapter <86> into monographs, the USP encourages manufacturers and vendors to submit data on recombinant testing across various sample types. This data will strengthen the case for including USP Chapter <86> in individual monographs, ultimately making recombinant technologies fully compendial and easier to implement.
Milestones in BET: From LAL prevalence to recombinant adoption
LAL has long been the gold standard for BET due to its compendial status, decades of data, and no reported cases of failure due to false negative tests. It is recognised as the primary endotoxin detection method in biologics by global standards, including the United States Pharmacopeia (USP), European Pharmacopeia (EP), and Japanese Pharmacopeia (JP).
Since the early 2000s, recombinant endotoxin testing methods have faced limited availability, with only one primary vendor offering these products. Despite rFC being introduced in 2003, its adoption remained minimal until recently, as more companies began recognising the benefits of alternative methods.
Having multiple suppliers has introduced a level of supply chain security that was previously unattainable
The recombinant methods lacked compendial recognition, requiring end users to undertake a full alternative method validation and regulatory submission, a complex and resource-intensive process guided by local regulators. While regulatory agencies have provided some guidance on method validation, there is still a grey area in terms of standardised 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 the investment with minimal regulatory acceptance, only to encounter roadblocks during review or face potential rejection altogether. As a result, pharmaceutical companies continued to show favouritism towards the safe option, LAL.
Additionally, validating alternative methods proved to be both costly and time-intensive. The introduction of rCR methods provided a solution by enabling end users to retain their existing equipment and processes. Yet, the challenge of validating alternative methods persisted.
Pharmaceutical companies needed to ensure that recombinant methods met the same rigorous standards for sensitivity, precision, reproducibility, and equivalency established for LAL.
The growing demand for alternative endotoxin testing methods
As recombinant technologies gained recognition as a more sustainable approach for bacterial endotoxin testing (BET), the industry began to shift.
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