USP <86> Bacterial Endotoxins Test Using Recombinant Reagents: Comparing rFC and rCR Technologies

Endotoxin testing is essential for ensuring the safety of injectable pharmaceuticals and medical devices. Traditionally based on the use of limulus amebocyte lysate (LAL) from horseshoe crab blood, the field is now embracing ethical and sustainable alternatives. With the introduction of USP <86>, the United States Pharmacopeia formally recognizes recombinant reagents for endotoxin detection, marking a turning point in laboratory testing practices.

In this article, we explore the scope of USP <86> Bacterial Endotoxins Test Using Recombinant Reagents: Comparing rFC and rCR Technologies.

What Is USP <86>?

USP <86> Bacterial Endotoxins Test Using Recombinant Reagents is a newly introduced chapter that outlines validated methods for using non-animal-derived recombinant reagents in bacterial endotoxin testing. This chapter complements USP <85>, which describes the classic LAL method.

By endorsing recombinant methods, USP <86> supports the global 3Rs initiative—Replacement, Reduction, and Refinement of animal testing—and provides a path for laboratories to maintain compliance while eliminating the use of animal-derived lysate.

Why Move Toward Recombinant Testing?

The shift from traditional LAL testing is driven by growing concerns over environmental sustainability, ethics, and test consistency. Harvesting horseshoe crab blood for LAL poses ecological risks, while recombinant methods offer animal-free, sustainable alternatives without compromising performance.

Recombinant technologies such as rFC and rCR improve reproducibility and reduce variability, making them ideal for critical quality control processes. With increasing regulatory support—including recognition in USP <86> and Ph. Eur.—these methods meet compliance needs while aligning with global efforts to reduce animal testing and support sustainable science.

Recombinant Factor C (rFC)

Recombinant Factor C isolates the endotoxin-specific component of the horseshoe crab’s immune cascade using recombinant DNA. When exposed to lipopolysaccharides (LPS), rFC generates a fluorescent signal proportional to the endotoxin concentration.

Key Benefits of rFC:

• No cross-reactivity with β-glucans

• Animal-free and sustainable

• Highly specific to endotoxins

• Excellent reproducibility and sensitivity

  
  

Lab Integration:

• Typically used in fluorescence-based assays

• May require new workflow setup

• Product suitability may be limited

Recombinant Cascade Reactions (rCR)

rCR technology replicates the entire LAL cascade using recombinant forms of Factor C, Factor B, and the pro-clotting enzyme. It is designed to mimic the traditional LAL assay as closely as possible.

Key Benefits of rCR:

• Integrates seamlessly with existing LAL lab workflows

• Compatible with turbidimetric and chromogenic formats

• Animal-free, meeting 3Rs compliance

• Suitable for wider range of finished products

  
  

Considerations:

• May cross-react with β-glucans, similar to LAL

• Slightly more complex cascade system

Bacterial Endotoxin Test: Comparing rFC and rCR Technologies

Feature	rFC	rCR
Cascade Components	Recombinant Factor C only	Factors C, B, and pro-clotting enzyme
Detection Mechanism	Fluorescence	Chromogenic or turbidimetric
Cross-reactivity with β-glucans	None	Possible (similar to LAL)
Workflow Compatibility	Requires new assay setup	Compatible with existing LAL workflows
Sustainability	Fully animal-free	Fully animal-free
Type of Assay	End point	Kinetic

The Future of Endotoxin Testing

The introduction of USP <86> validates the scientific and regulatory viability of animal-free endotoxin testing. Both rFC and rCR offer powerful, compliant alternatives to traditional LAL methods.

Whether your lab prioritizes workflow continuity (rCR) or maximum specificity (rFC), these recombinant technologies provide robust options for ensuring the safety of sterile pharmaceutical and medical products—without compromising ethics or performance.

Reference

USP <86> Bacterial Endotoxins Test Using Recombinant Reagents