Rabbits, Horseshoe Crabs and Now Recombinants

Rabbits, Horseshoe Crabs and Now Recombinants: The Exciting Journey of the Endotoxin Testing Industry

From the rabbit pyrogen test (RPT) to the Limulus Amebocyte Lysate (LAL) method and now the recombinant Factor C (rFC) assay – the endotoxin testing industry has been on an exciting journey! And as Lonza’s recent annual Global Endotoxin Testing Summit demonstrated, there is still so much more to come. Not least because of the pioneering rFC test, which shows great promise and is increasingly being recognized by regulatory authorities, including the European and Chinese Pharmacopoeias, as an alternative, sustainable method.

As a fascinating forum for endotoxin testing scientists and end-users to get together and discuss the latest developments in the field, the summit explored the evolution of detection methods that already spans more than 50 years. The challenges associated with Low Endotoxin Recovery (LER) were also addressed. Keep reading to learn more from the perspectives and experiences of the summit speakers.

The LAL Test: A 50-Year Impeccable Safety Record

Bacterial endotoxin (the lipopolysaccharide (LPS) component of the outer cell membrane of Gram-negative bacteria) is everywhere. Bacterial endotoxins are enormously powerful biological reagents that, if they enter the bloodstream, can cause various pathophysiological reactions by triggering the major physiological systems in the human body which are based on a cascade mechanism. As such, it is vital that parenteral drug products and medical devices do not contain clinically significant levels of bacterial endotoxins before being released to the market.

In 1968, Professor Jack Levin, M.D., University of California School of Medicine, San Francisco, and the late Professor Frederick B. Bang, M.D., Johns Hopkins University School of Hygiene and Public Health, Baltimore, described how bacterial endotoxins stimulate the coagu¬lation cascade contained in the cytoplasmic granules of amebocytes which circulate in the blue blood of horseshoe crabs. This enzymatic reaction, initiated by activation of Factor C by bacterial endotoxin, forms a coagulin clot, the rate of appearance of which is related to the concentration of bacterial endotoxins present in the sample. This ‘accidental’ discovery led to the creation of the Limulus Amebocyte Lysate (LAL) test, which subsequently officially replaced the Rabbit Pyrogen Test (RPT) for testing parenteral pharmaceuticals and medical devices for fever-inducing bacterial endotoxins.

“Before the LAL assay was introduced, only the Rabbit Pyrogen Test was available for the detection of fever-causing bacterial endotoxins,” explained Professor Levin during his keynote presentation at this year’s summit. “Over the years, the method’s applications have increased enormously, for example testing of short-lived radioisotopes, plasma protein fractions, meat products and water supplies. However, I find it very frustrating that it has not been certified yet for cerebrospinal fluid testing. Although the LAL test cannot identify specific organisms causing meningitis, it can classify them as Gram-positive or Gram-negative, enabling physicians to quickly decide what type of antibiotics to initially administer while waiting for the culture results.”

Nevertheless, with sustainability being at the top of the agenda across the endotoxin testing industry, much pressure is being applied to establish an animal-free method. Recent validation and study data by Eli Lilly support that the sensitivity of the rFC test is similar to that of the LAL method.

Unlocking the Power of Recombinant Factor C

rFC is the recombinant version of the natural Factor C protein found in horseshoe crab blood. Although the method was originally faced with scepticism, it is now increasingly being recognized as biochemically equivalent to the LAL assay, delivering accurate, specific and consistent results without the dependency on horseshoe crab blood for obtaining the lysate source material. At the summit, Mr. Jay Bolden, Senior Consultant Biologist, Eli Lilly & Company, presented data demonstrating that the rFC and rLAL assays comparably detect endotoxins in the same model system as the LAL test in a diverse set of pharmaceutical products, and in environmental contamination models.

“In addition to the sustainability advantage of the rFC method, we’ve been able to establish that the rFC and rLAL assays are insensitive to the false-positive Factor G pathway to which commercial LAL tests are susceptible, and which could have a negative impact on accuracy of results,” noted Bolden. “Importantly, given that the rFC assay is produced through a standardized, validated manufacturing process, its widespread adoption could facilitate analytical consistency across different laboratories.”

Supply chain security is an additional benefit enabled when using the rFC method. As discussed in our previous blog, the increased horseshoe crab mortality in Asia will inevitably increase the reliance of the biomedical industry on the North American horseshoe crab species as the main source for producing LAL assays. The rFC test can effectively alleviate this problem.

“Given these unique benefits, a natural progression would be for the U.S. pharmacopoeia (USP) to revise its harmonized bacterial endotoxin testing (BET) chapters to include rFC as a technology equivalent to LAL,” said Bolden. “I was really excited to present new data at this year’s summit to make a stronger argument towards that direction. Over the last year, rFC has attracted a lot of attention across the biopharmaceutical industry and we can safely predict that most of the big companies will convert to this sustainable technology within the next five to 10 years.”

“For conservation reasons, it is important that the industry continues the uptake of rFC and the method becomes USP-accepted,” added Mr. Kevin Williams, U.S. Endotoxin Senior Scientist, Hyglos, a BioMerieux Company, while offering key insights into the basics of bacterial endotoxins. “I’ve used the LAL assay for 30 years, so I know how effective and reliable the test is. This is a matter of bridging the old with the new to adopt a technology that ensures the safety of parenteral pharmaceuticals and medical devices, while also protecting the global ecosystem.”

However, whether using the LAL or the rFC method, LER remains a key consideration as it could potentially affect the accuracy and sensitivity of endotoxin testing.

LER Mitigation Strategies: Why a One-Size-Fits-All Approach Can’t Work

The LER phenomenon is widely thought to be initiated by a change in the micellar supramolecular structure of the LPS, in which the lipid A moiety is less accessible. Given the potential negative impact of LER on the reliability of endotoxin testing, much focus has been placed on identifying effective strategies for mitigating test interference and endotoxin masking. According to the new Technical Report No. 82 (TR82) on LER published in March 2019 by the Parenteral Drug Association (PDA), the goal of mitigation is to manage situations where LER is observed by BET in hold-time studies in a manner that minimizes the risk of undetected endotoxin in drug products.

The key to LER mitigation is to facilitate the formation of detectable LPS structures. This can be achieved by enabling destabilization or swelling of the masking complex, or suppressing micelle formation. While addressing this issue at the summit, Dr. Ruth Röder, Project Leader of Endotoxin Services, Microcoat Biotechnologie GmbH, made it explicit that, “LER mitigation strategies have to be developed on a case-by-case basis. There is no universal approach that could be successfully applied on all occasions.”

Dr. Röder explained that, when developing a mitigation strategy, it is essential to analyze the sample under investigation to identify what could possibly lead to the LER effect. Equally important is to understand the underlying mechanism of LER. Using this combined knowledge, it becomes easier to choose the most suitable mitigation strategy from a large selection of options, including sample dilution, elimination of the masking protein by digestion, and neutralization of masking components through addition of demasking components. Whatever the method of choice, the development of demasking protocols to address the LER phenomenon can be particularly time-consuming, slowing down the release of new products until the right strategy has been identified.

“In my opinion, LER will be an ongoing challenge for the biopharmaceutical industry, with each sample requiring the development of a custom demasking strategy, concluded Dr. Röder. “Given that new samples, such as cell and gene therapies, will keep emerging, new extensive research projects will be needed to introduce novel mitigation approaches. Hopefully, gaining more knowledge about the underlying mechanism of the masking effect and how it can be addressed will eventually ease and accelerate this process.”

Taking an All-Inclusive Approach to Endotoxin Testing

With the safety of parenteral pharmaceuticals and medical devices being an utmost priority, it is important that biopharmaceutical manufacturers carefully and thoroughly evaluate all available endotoxin testing technologies. In order to choose an appropriate test method, it is necessary to understand the formulation of the drug product in development and the manufacturing process it needs to go through.

It is also advisable to fully review and use all available knowledge and resources. Professor Levin stressed how the new PDA TR82 on LER has collected and synthesized an enormous amount of information and includes a comprehensive reference list. “Anyone performing bacterial endotoxin testing studies should have this document in hand,” he said.

Finally, it is critical to ensure that the chosen endotoxin testing technology has a positive impact on the environment. However, as Williams pointed out, endotoxin testing researchers and end-users still have a long way to go to reach consensus around the key challenges facing the industry, such as adopting a sustainable detection technology and addressing the LER phenomenon. It is important to keep an open mind and recognize that all parties have legitimate concerns and reasons to support specific approaches.

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