A new chapter in Quality Control – how USP Chapter <86> can transform approaches to endotoxin testing

Endotoxins, cell wall components of Gram-negative bacteria, pose a significant risk to patient safety. Since endotoxins can cause severe inflammatory responses including fever, septic shock and even death, their content must be rigorously tested and controlled in injectable products and Medical Devices as well as in water for injection (WFI) raw materials and primary containers used for production. They are very stable and heat resistant and cannot be easily removed by filtration. The only effective strategy is prevention and monitoring through accurate methods during manufacturing process and before product release.

Market research suggests that between 70 million and 100 million endotoxin tests are carried out each year – at this level of volume, consideration should also be taken as to the environmental impact and sustainability of these tests. Traditionally, endotoxin testing was performed through the use of LAL reagent, extracted from Horseshoe Crabs. The pharmaceutical industry, however, is facing a challenge now: “Can we keep relying on an animal-based system for our supply chain, when horseshoe crabs are endangered, the number of tests per year is growing fast and science has evolved thanks to recombinant DNA technologies?” That's how and why recombinant BET methods were born. While the majority of endotoxin testing is still carried out using traditional methods, the emergence of new recombinant reagents creates an opportunity to weigh up the benefits of a potential shift. Manufacturers evaluating their current approach to endotoxin testing and considering a switch to modern testing methods may wish to reflect on the following key factors:

1. Reliability

rFC agents are produced to only contain the specific component of LAL that is activated by the presence of endotoxins, increasing batch-to-batch consistency vs animal sources. In another field of reliability, recombinant agents also offer a more stable production source than LAL, as they can be produced when needed rather than manufacturers being reliant on their seasonal availability.  

2. Ethical considerations

The pharmaceutical industry’s reliance on LAL for endotoxin testing raises ethical challenges for manufacturers because LAL itself is derived from the blood of horseshoe crabs living in the wild. While it’s true that historically the manufacture and testing of sterile products has relied on animals, these practices have been in decline for some time and principles such as the 3Rs have seen companies’ look to replace, reduce and refine their use of animals. In the case of endotoxin testing, switching from LAL to recombinant agents could save 90,000 animals per year,¹ providing another powerful argument for moving away from traditional methods.

3. Efficiency

Another considerable benefit offered by endotoxin testing with recombinant reagents is improved efficiency. Traditional LAL testing often requires multiple steps – multiple pipetting, curve-creation, daily lysate reconstitution and cost-loss due to waste of lysate… When using an rFC assay, 21 results can be obtained in 30 minutes at a sensitivity of 0.05 EU/ml, or in 1 hour at 0.005 EU/ml. The use of pre-coated plates eliminates the most time-consuming and error-prone steps, while preserving the sensitivity and compliance required in pharmaceutical QC.

This approach also helps optimize lab operations by reducing operator burden, thanks to shorter training time, fewer manual steps, and minimal troubleshooting, freeing up technician capacity and controlling labor costs. Robust performance across users and shifts lowers the risk of invalid results, minimizing retests and disruptions to daily lab planning. Testing with rFC reagents also enables the easy integration of automated processes, which can generate further efficiencies as well as reducing the potential for human error.