How recombinant LAL reagents differ from each other?

Published on 1st September 2025

Commercially available recombinant reagents now come in two types: Recombinant Factor C (rFC) and Recombinant Cascade Reagent (rCR). The US, Japanese, and European Pharmacopoeia monographs accept both rFC and rCR methods as LAL alternatives, but application-based suitability testing is still required.

 

Pharmaceutical products, including vaccines, can be contaminated by endotoxins, which are heat-stable lipopolysaccharides from Gram-negative bacteria. The pyrogenic properties of endotoxins necessitate endotoxin testing for parenteral pharmaceutical products to prevent severe physiological reactions in patients. Currently, the prevalent method for testing is the animal-derived Limulus Amoebocyte Lysate (LAL) assay. However, assays using recombinant factors, a reagent not derived from animals, have been suggested as alternatives [1] and proposed as methods for compendial acceptance [2].
Commercially available recombinant reagents now come in two types:

  • Recombinant Factor C (rFC)
  • Recombinant Cascade Reagent (rCR).

While the Japanese Pharmacopoeia, US Pharmacopoeia, and European Pharmacopoeia monographs accept both rFC and rCR methods as LAL alternatives, application-based suitability testing is still required [2]. 

Recombinant Factor C (rFC) 

The European Pharmacopoeia and the United States Pharmacopeia recognise rFC assays as an official method. Recombinant Factor C (rFC) reagents consist solely of the recombinant Factor C component of the coagulation cascade. This testing uses an endpoint fluorometric method [2]. The methods described in Ph.Eur 2.6.32 and USP <86> for rFC is the description for this endpoint flourescent method. 

Recombinant Cascade Reagent (rCR) 

The US Pharmacopia recognises rCR as an official method. The cascade reaction of three protease zymogens and a coagulogen is the foundation of the molecular mechanism for haemolymph coagulation by endotoxin. The cascade begins with the activation of the zymogen (factor C) by the endotoxin. Factor C's activation triggers the activation of factor B.
Factor B, in addition to its known role in the coagulation cascade (transmitting signals from factor C), also binds to endotoxin. The stepwise activation amplifies signals, enhancing sensitivity. The protease activity of the rCR, containing recombinant factor C, is significantly higher than that of recombinant factor C alone, demonstrating the substantial signal amplification achieved through the cascade reaction.
The proclotting enzyme is the third enzyme added that comes from the genome of Limulus polyphemus.
A chromophore is added that is activated by the clotting enzyme, just as it is apart of chromogenic LAL reagent.
The rCR testing methodology parallels the photometric quantitative (kinetic chromogenic) techniques outlined in USP <85>. The ability to run kinetic measurements allows for lower sensitivities to be measured, equivalent to the KCA reagents in USP <85>. Kinetic measurements typically allow for a finer sensitivity and wider quantification range as they can accommodate the multiple different activation times of many standard concentrations. 

PYROSTAR™ Neo +: A Superior rCR Based Alternative

Recombinant proteins offer a sustainable, animal-free method for mass production, eliminating ethical concerns and scaling limitations [3].
Our new PYROSTAR™ Neo+ reagent, a recombinant protein, uses the Kinetic Chromogenic Assay (KCA) to detect gram-negative bacterial endotoxin levels. This reagent is a comprehensive solution for LAL assays, incorporating recombinant Factor C and Factor B, the pro-clotting enzyme (essential LAL components), a chromogenic substrate, and a suitable buffer solution. A chromogenic substrate allows for the quantitative measurement of endotoxin levels, mirroring the process of the traditional LAL method and providing a precise numerical result. Furthermore, the PYROSTAR Neo+ system demonstrates a decrease in interference from certain medications and exhibits heightened sensitivity to naturally occurring endotoxins, thus broadening its applicability to a wider array of samples and uses, such as evaluating product safety and analysing pharmaceutical materials and the water used in their production.

 

References

  1. Marius, M., Vacher, F. & Bonnevay, T. (2020). Comparison of LAL and recombinant Factor C endotoxin testing assays in human vaccines with complex matrices. PDA Journal of Pharmaceutical Science and Technology, 74: pdajpst.2019.010389.
  2. USP (2025). Chapter <86> Bacterial Endotoxins Test using Recombinant Reagents.
  3. Kang, D.H., Yun, S.Y., Eum, S., Yoon, K.E., Ryu, S.R., Lee, C., et al. (2024). A Study on the Application of Recombinant Factor C (rFC) Assay Using Biopharmaceuticals. Microorganisms, 12(3): 516.