Following on from our previous meetings, rFC was once more discussed as an alternative to LAL, as was the potential of pyrogen detection using a cryopreserved peripheral blood mononuclear cell (PBMC)-based monocyte activation test (MAT) assay. Attendees also had the chance to delve into the new Technical Report No. 82 (TR82) on low endotoxin recovery (LER) published in March 2019 by the Parenteral Drug Association (PDA). Together they learned about effective strategies for mitigating LER interferences sometimes associated with the LAL test. Automation and data integrity were discussed during the event too, providing a fully rounded program.
Q: Why is sustainable endotoxin testing a necessity?
A: Horseshoe crabs play a vital role in the global ecosystem, not least because their eggs are a key source of food for several species of shorebirds, including red knots, helping them to survive annual migratory journeys of up to 14,000 km. In the late 1980s-early 1990s, the population of horseshoe crabs dropped considerably due to overfishing for bait in eel and conch fisheries. In response, the Atlantic States Marine Fisheries Commission (ASMFC) introduced the 1998 Interstate Fishery Management Plan for Horseshoe Crab, which established state-by-state quotas in all Atlantic states for horseshoe crabs harvested for bait.
As there is an unquestionable necessity for the biomedical industry to keep producing new, safe drugs, the ASMFC cannot impose a hard quota on horseshoe crab fishing for biomedical applications. However, both the ASMFC and the ERDG actively encourage the industry to adopt alternative, environmentally friendly methods, although it’s important to note that the horseshoe crabs are not harmed during the blood collection process and are safely released back into the sea.
As the Chairman of the ASMFC Horseshoe Crab Advisory Panel while also representing the biomedical community of the state of Maryland, I need to make sure the industry’s best interests are served while minimizing the impact on these ancient species and the wider ecosystem. That’s where rFC comes into play, and summit attendees had a unique opportunity to hear all about the latest developments surrounding this innovative technology.
Q: How does recombinant Factor C promote sustainable endotoxin testing?
A: The rFC assay is based on a synthetic equivalent of Factor C, which is the first component of the horseshoe crab clotting cascade that is activated in response to endotoxins. By adopting this sustainable, animal-free method, the biomedical industry can play a key role in the conservation of the world’s horseshoe crab species.
A recent ASMFC report demonstrates that the harvest of horseshoe crab blood for biomedical use does not negatively affect the populations of the Atlantic horseshoe crabs. It is a different situation in Asia, though, with increased crab mortality due to overfishing, habitat loss, their use for human consumption and because they are not normally returned to the sea following the bleeding process. Inevitably, this will increase the reliance of the biomedical industry on the North American horseshoe crab species as the main source for preparing LAL-based assays.
Not only can the rFC method help alleviate this challenge and reduce the pressure on the horseshoe crab population, but as demonstrated at last year’s summit, the assay is as specific, sensitive and robust as the LAL method. This year’s event follows the recent approval for release by the U.S. Food and Drug Administration (FDA) of Eli Lilly’s EmgalityTM (galcanezumab), the first drug to have been validated using the rFC assay instead of the LAL test. This marks a significant breakthrough in establishing rFC as the non-animal method of choice for the identification of bacterial endotoxins in pharmaceutical products.
It is also important to note that the horseshoe crab blood can only be collected for a very short period of time every year, from the second week of June till mid-October in Maryland, limiting its supply considerably. Obviously, this is not an issue with rFC, as the method does not rely on these, or any other, animals for the source materials.
Q: Could you explain what the new PDA Technical Report means for the endotoxin testing industry?
A: LAL test interference and endotoxin masking were first observed back in the 1970s, but the term LER was first used in 2013 by Dr. Joseph Chen and Dr. Anders Vinther from Genentech. LER is now widely defined as the inability to recover >50% activity over time when highly purified endotoxin is added to an undiluted product.
Due to the potential concerns around patient safety, the PDA published the new Technical Report to provide guidance on developing robust and scientifically sound LER hold-time studies and to outline various mitigation strategies, such as the addition of dispersants and other sample treatments. Developed by subject-matter experts from academia, the U.S. FDA, the pharmaceutical industry, reagent/testing vendors and consulting firms, the Technical Report also includes scientific findings on the LER mechanism, useful data on clinical relevance and data from 12 case studies.
Together with other members of the PDA LER taskforce, we reviewed an extensive collection of documents, including forty years of Morbidity and Mortality Weekly from the Centers for Disease Control and Prevention (CDC) and adverse event (AE) reports from the FDA. It’s definitely reassuring that there has never been an incidence of a LAL test missing a pyrogenic pharmaceutical product, which due to endotoxin contamination could cause an endotoxin-induced reaction in the public, Nonetheless, companies must be made aware of potential product testing interferences, and know how to appropriately deal with them.
Q: What’s in the future for the endotoxin testing industry?
A: I’m hopeful that rFC will ultimately be recognized by the endotoxin testing industry as a method that’s as accurate, sensitive and specific as the LAL assay. In recent years, much progress has been made towards that goal. The European Pharmacopoeia is currently working on a standalone chapter on using rFC for bacterial endotoxin testing, and in January 2019, rFC was listed and described as a new compendia method for bacterial endotoxin testing in the Chinese Pharmacopeia. The 4th version of the Chinese Pharmacopeia will be effective in 2020. The United States Pharmacopeia has announced that a draft chapter for rFC testing will be published at the end of 2019.
The Biotechnology Innovation Organization (BIO) has set up a taskforce with members from the Unites States Pharmacopoeia, the U.S. FDA, reagent manufacturers and the pharmaceutical industry, and they plan to run a multi-center ring trial on rFC, the results of which are expected to be published in the next three to five years. Additionally, in 2017, a team of Japanese scientists published study results that support the use of recombinant reagents for bacterial endotoxin testing.
Couple these efforts with the approval of Eli Lilly’s EmgalityTM, and more pharmaceutical companies may consider adopting rFC in their laboratories. The added benefit is that rFC can be made anytime of the year in our cGMP-compliant manufacturing facilities and has the potential to replace the LAL test. We started this adventure back in 2003, and we really believe in the power of this innovative technology. Plus, we’ve got the data to counteract any doubts.
In addition to rFC, presentations from this year’s summit showed that automation, data integrity and MAT continue to be key themes. Check this space for our next blog post that will discuss the evolution from the LAL test to the rFC method, as presented at this year’s event.