Frequently Asked Questions for Bacterial Endotoxins Testing

Why is endotoxin testing required for pharmaceuticals?

Endotoxin testing ensures product safety by detecting harmful bacterial components that can cause severe immune reactions. This quality control procedure is mandatory across pharmaceutical, medical device, and biotech industries for products contacting human blood or body fluids - including injectable drugs (parenteral drugs) like vaccines and insulin, as well as medical devices such as catheters, implants, and dialysis equipment. Water for injection (WFI) and other pharmaceutical water systems also require regular endotoxin monitoring as they're critical raw materials in drug manufacturing. Even trace amounts of endotoxins can be dangerous, making testing essential for patient safety and regulatory compliance.

What is the LAL bacterial endotoxin test procedure?

The LAL (Limulus amoebocyte lysate) test has been FDA-approved for 40 years as an in vitro alternative to rabbit pyrogen testing. Using horseshoe crab blood lysate, it detects bacterial endotoxins through three methods: gel-clot (qualitative), turbidimetric, and chromogenic (both quantitative). Results are measured via gel formation, turbidity, or color change against standard curves. Testing requirements are outlined in USP <85>, EP 2.6.14, JP 4.01, and ChP 1143, with new recombinant methods (using rFC or rCR) now available.

What is the US Pharmacopoeia guidance on recombinant BET methods?

USP Chapter <86> "Bacterial Endotoxins Test Using Recombinant Reagents" became official in May 2025. USP <86> provides additional techniques for endotoxin testing using non-animal derived reagents, specifically the recombinant Factor C (rFC) protein or a recombinant cascade reagent (rCR). These methods offer additional techniques to the current bacterial endotoxins tests described in USP <85>.

USP Chapter <86> describes how users can use traditional LAL or recombinant reagents for endotoxin testing by leveraging the recombinant reagent's Primary Validation Package to demonstrate the method is suitable for its intended use. With the publication of this chapter and the use of the Sievers Eclipse, users can achieve results in significantly less time than with traditional methods and with a greater emphasis on sustainability. The plate set up takes approximately 5 minutes using the Sievers Eclipse, including the standard curve, and uses up to 90% less reagent.

What are barriers to the use of recombinant bacterial endotoxins test methods, and how can any hurdles be streamlined for labs?

Prior to adopting recombinant reagents such as rFC or rCR, the user should first evaluate the reagent manufacturer's Primary Validation Package with their Quality team to determine whether there are any gaps in testing that need to be remedied prior to adoption. Similar to adopting any new method, it is a normal requirement to demonstrate that the method is fit for use. The use of recombinant reagents is still considered to be an alternative method, so users must prove that the reagent is suitable for its intended purpose under the conditions of use for the material, drug substance, and/or drug product. If there are gaps with the Primary Validation testing of the reagent, further testing may be required. Additional testing can be done with autochthonous organisms that are commonly present in the manufacturer's facility, as well as purified endotoxin standards.

A side-by-side protocol or a comparability study comparing current methods to rCR reagents will provide the necessary data to demonstrate comparability. While transitioning existing products to rCR may seem challenging, analysts only need to complete alternative method testing followed by a single-lot verification to confirm the rCR reagent performs similarly to existing methods like chromogenic assays.

A microfluidic platform like the Eclipse streamlines method validation through rapid assay setup. With integrated liquid handling plus embedded endotoxin standards and PPCs, assays can be configured in minutes using LAL or rCR.

What are endotoxins and why are they dangerous?

Endotoxins are lipopolysaccharide components from gram-negative bacterial cell walls, specifically the Lipid A portion that triggers fever and immune responses. These potent toxins become life-threatening when present in bloodstream-contacting products above safe concentrations. They can contaminate pharmaceutical water systems, raw materials, and finished products throughout the manufacturing process. Water systems are particularly susceptible to endotoxin contamination as bacteria can form biofilms in pipes and storage tanks. Global regulators including the FDA mandate endotoxin testing for all life sciences products due to their severe impact on patient safety. Compendial test methods for pharmaceutical waters include requirements for bacterial endotoxin limits in waters for parenteral (injectable) use, such as Water for Injection and Sterile Water for Injection.