Recombinant Technology for Bacterial Endotoxin Testing: What’s Hot in 2022
A synthetic alternative to Horseshoe Crab blood is being considered but are they as good as the natural LAL?
For over a decade, companies have been working on recombinant proteins that mimic those found naturally in the remarkable blue blood of the Atlantic Horseshoe Crab for the detection of bacterial endotoxins.
Bacterial endotoxin testing (BET) is a mandatory safety requirement in the pharmaceutical and biomedical industries. Bacterial Endotoxin Testing is required on all batches of injectable drug products and implantable devices before they can be released for use in patients. In addition to the end product, the raw materials, intermediates, and excipients for these processes are also tested as part of the quality control manufacturing process.
Approaching 75 million tests per year, the majority of bacterial endotoxin tests are carried out via the Limulus Amoebocyte Lysate (LAL) assay, which is derived from the blood of the Atlantic Horseshoe Crab (HSC). In South Carolina, these crabs are hand collected once per year in the spring months when they emerge from the ocean to spawn. They are carefully handled and transported, following strict procedures, to act as blood donors, providing a small amount of blood per animal, and then returned to their spawning spots within 24 hours.
As technology advances, a synthetic alternative to the Horseshoe Crab blood is being considered for this critical safety test. Known as recombinant reagents, the main question in the industry remains, “are they as good as the natural LAL that has been proven reliable for over 50 years?” Our testing, as well as other reputable organizations, shows that single protein reagents can potentially underestimate, or miss detecting the natural endotoxins found in the environment in comparison to the traditional LAL test. Further studies using the original Rabbit Pyrogen Test also indicate that single protein reagents lose the sensitivity to environmental endotoxins that LAL offers, which poses a potential patient safety risk.
Multi-protein recombinant reagents also have their drawbacks, and it is clear that formulation of any recombinant reagent has a huge impact in its ability to recover endotoxins. While the industry relied on Reference Standard Endotoxin in the past to standardise manufacturing of LAL reagents, this may not be enough when manufacturing recombinant reagents and new standards may need to be established.
Fortunately, great progress is being made in this area. Our scientists at Charles River Microbial Solutions are researching, testing, and understanding the multi-protein reagent formulations (full cascade including Recombinant Factors C, B and Pro-Clotting Enzyme). We have a formulation that may be comparable with LAL and offer the safety required. More time is required by our labs and the other commercial developers to understand these reagents further and gather more data before they become routinely used in QC testing.
Fifteen years of side-by-side experiments had to be conducted to ensure product safety and efficacy of LAL as compared to the rabbit pyrogen test to gain FDA approval. Fast forward to today; although we have advanced in our technology and innovation, ample and diverse testing needs to be conducted to ensure the same product safety and efficacy of recombinant reagents to LAL. The safety of every single injectable is dependent on LAL, and the remarkable animal who provides it to us.
2022 may not be the year of the recombinant LAL product, but look for intensified efforts to resolve questions about their safety and more guidance from regulators.
- Nicola Reid, Associate Director, Product Management, Endotoxin Products, Charles River
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