How much risk are you willing to take in the safety of your product and patients? Why the alternative endotoxin detection assays should make industry pause.

About 40 years ago, the ancient horseshoe crab, which dates back at least 440 million years, assumed a new role. After researchers discovered that its blue blood contained a vital protein capable of detecting harmful endotoxins found on the outer membrane of Gram-negative bacteria, this species became a vital partner in fighting the spread of infection. Every drug or device approved for parenteral use, worldwide, must be evaluated using the bacterial endotoxin test (BET) for the potential to induce a fever in a patient (pyrogenicity).Today there are approximately 70 million Limulus Amebocyte Lysate (LAL) tests performed annually that are derived from horseshoe crab blood. Pharmaceutical and medical device manufacturers use the test to ensure their products remain endotoxin-free and that their patients remain safe.

Currently, the verified, FDA-approved standard for the BET is the LAL assay, which is produced by concentrating and lysing the blood of the Atlantic horseshoe crab. There are some, both in and outside of the pharmaceutical sector, who believe that the supply of LAL may not be able to keep pace with this growing demand. Citing this presumption, along with concerns that purports to eliminate the need for animal-derived products for bacterial endotoxin testing, alternative endotoxin detection assays have been developed. While this could ostensibly be a way to reduce substantially our reliance on horseshoe crabs, the synthetic products carry their own sets of challenges. These methods promise laboratory-based, rapid, specific alternatives to the standard LAL assay. However, not only do these alternative methods require extensive validation, but according to recently published data legitimate concerns also exist regarding the accuracy, specificity, robustness, and reproducibility of these assays.  

Moreover, the synthetic tests could, ironically, leave the horseshoe crabs vulnerable to the bait industry. Alan Hoffmeister, Senior Endosafe Product Specialist for the Microbial Solutions division of Charles River, which is an FDA-licensed LAL manufacturer, discusses some of the major differences between the recombinant factor C and LAL tests and why pharmaceutical and medical device companies might want to be cautious about using them. 

Validation is obviously key for any new product. How much data is publicly available about the reliability of synthetic products such as recombinant C?         

AH: Validation is a major component for any new testing method that is looking to be adopted. Without a scientifically sound validation, the reliability of the new method cannot be assured. All compendial methods must be validated in accordance with scientifically accepted principles, and this would often include independent multi-lab collaborative studies, designed to assure that the new method does indeed meet the validation requirements. LAL, for instance, underwent years of testing before it was deemed suitable for use as an end-product release method for drug products and medical devices.  That testing included side-by-side testing against the then-only compendial method available, the rabbit pyrogen test (RPT). Over a number of years, hundreds of thousands of LAL results were compared against tens of thousands of RPT, and these tests proved LAL to be more sensitive, reliable and reproducible than RPT. It was only after such exhaustive testing that LAL was officially accepted as a suitable replacement to RPT. As of today, rFC has not faced such scrutiny, and therefore there is still doubt as to its reliability. Much of the data that does exist was either produced by the manufacturers themselves, or commissioned by them, and therefore may be subject to a conflict of interest. As a result, currently firms wishing to adopt rFC, as an alternative to LAL are required to carry out their own alternative methods validation, where they test for Specificity, Linearity, Range, Accuracy, Precision, Limit of Detection, Quantitation Limit and Robustness. Without this rFC cannot be used to replace the LAL assay for end product release. But still this is nowhere near the level of scrutiny that LAL underwent, and as such its reliability must still be questioned.

What are the regulatory requirements for recombinant products and LAL tests?

AH: Back in the early 2000s, the US, European and Japanese Pharmacopeia harmonized their mandatory chapters on bacterial endotoxin testing (BET). The chapters specify that endotoxin tests must use LAL or Tachypleus Amebocyte Lysate (TAL) reagent, manufactured in accordance with the requirements of the competent authority, which in the case of LAL would be the US FDA. Since recombinant products don’t take Amebocyte from horseshoe crab blood, therefore they are not LAL or TAL, and as such they don’t comply with this requirement. In addition they do not need to be manufactured to FDA requirements and standards. Instead, companies wishing to use these recombinant products must perform their own alternative methods validation, and must seek prior approval to use the alternative method from the respective competent authorities, for each drug or medical device they wish to release with the alternative method. Alternative methods validation must include specificity testing, to prove the method capable of detecting endotoxin from all relevant sources. This would entail testing endotoxin from a variety of Gram negative organisms, including organisms isolated from the natural flora of each company site performing the validation. The number of organisms included in this testing must be sufficient to prove specificity and can be a daunting challenge for even the largest of organizations.  

This lack of regulation can’t be good news for pharmaceutical companies or patients, correct?

AH: No, it’s a huge risk. There are literally hundreds of gram-negative bacteria species and you don’t necessarily know which is going to be the source of the endotoxin which could be in your product. I was at a conference a few years ago, and there was an MHRA inspector giving a talk. He said that when he audited companies, one of the things he would do was to apply what he called “the son or daughter test”—that is would he be happy with a product manufactured by that Company to be injected into his son or daughter. If he couldn’t answer unequivocally yes to that question, then he knew there was a problem. And it’s not just human safety, by the way. These products are also being used to treat animals, so animal lives are at risk as well.

What about LAL? Do we also encounter false-negatives?

AH: There have been no reported endotoxin outbreaks that have been traced back to a failure of the LAL test. There have been endotoxin episodes attributed to certain products, but in each of these cases the failure has not been due to the LAL assay missing endotoxin, but rather failures by the company’s testing procedures or a change to the products dosing regimen. The last major incident, reported in the New England Journal of Medicine in 1999, involved the use of the antibiotic, gentamicin in the US. The physicians who were administering it decided rather than follow the labelled dosing regimen of three doses spread over a 24 hour period, they would combine the three into a single injection. Unfortunately, they didn’t take into account that endotoxin limits are based on the maximum dose that can be given in a single one-hour period. When they combined the three doses they also combined the endotoxin they contained which then exceeded the endotoxin limit. It led to the United States Pharmacopeia having to reduce the endotoxin limit for gentamicin by two thirds, to take into account the increased dose.

Are synthetic products bound by the same FDA scrutiny as LAL?

AH: No, that’s again one of the issues. Because it was being used to replace the rabbit pyrogen test and because it is derived from blood—in this case from the horseshoe crab—the FDA decided LAL reagents should be monitored and licensed, so you have to be a licensed approved manufacturer to produce LAL. But that is all the FDA licenses. Every other product we produce, our endotoxin, water, buffers, software, instrumentation, none of that is licensed. The reason the FDA does not license recombinant products is that they do not come from the blood of the horseshoe crab. That means they don’t have oversight over the manufacturers of these products. This contrasts with licensed LAL manufacturers like us, who are audited just like a pharmaceutical drug manufacturer. We get unannounced inspections and we have to meet all of the requirements for the manufacturer of this product including current Good Manufacturing Practices (cGMP’s). This ensures the quality of LAL is maintained, and our customers can rely on it to release safe and effective drug products. By contrast, there is no FDA oversight for the manufacture or quality of the recombinant products. Any manufacturer can set up and produce rFC without needing to meet any regulatory requirements. There is no regulatory oversight as to how they manufacture or assess the quality of the reagents they produce. This presents a risk to those companies using these unregulated products, and to the patients receiving products released with these reagents.

Could it also be damaging for a company’s reputation to miss endotoxin from a gram-negative organism and release a contaminated product?

AH:  It could be absolutely devastating. Look at what happened a few years ago with that compounding pharmacy in New England. They were held responsible for producing contaminated drugs that killed 64 and harmed over 750 people across the country. The company no longer exists and the owner and several employees were jailed. Similarly, it would be catastrophic if a company released a drug product that killed patients, and it was later determined that endotoxin had not been picked up by the system they were using to detect them.

Has there been any studies that show this?

AH: Yes, a white paper co-authored by a supplier of alternate endotoxin methods, compared two LAL reagents to three alternate endotoxin detection methods for their ability to detect naturally occurring endotoxins1. Two of the alternate rFC methods were direct assays; the third method utilized a phage ligand, which is purportedly specific for bacterial endotoxins. The authors report that all the recombinant methods demonstrated a 94.4% correlation. But what about the 5.6% that did not agree? Interestingly, a comparison of LAL and rFC from the same supplier revealed two water samples where rFC under-predicted endotoxin contamination by 80% and 91% respectively. In one instance, the endotoxin concentration measured by the phage ligand was approximately six times greater than both of the direct rFC methods. No attempt was made to ensure that specificity was thoroughly resolved. In my opinion, this is an unacceptable patient safety risk for many.

There seems to be a push for a 3R’s initiative, how do current endotoxins methods play into this approach? 

AH: The FDA-licensed LAL cartridge, which uses only 1/20th the amount of traditional LAL reagents is one way we are reducing our reliance on horseshoe crabs. Potentially, if everybody performing an LAL test switches to cartridge, Charles River on its own could provide enough tests to supply all of the global testing requirements with fewer crabs needing to be bleed. On the other side of the coin, when horseshoe crabs weren’t needed for biomedical research, they were sacrificed by fisherman for commercial bait. So they were caught, quartered, put in bait pots and used to catch eel. Solely because of the biomedical industry, that practice stopped in South Carolina completely.

References

  1. 2.6.30. Monocyte-Activation Test. In European Pharmacopoeia; Council of Europe: Strasbourg: Council of Europe.

For more information surrounding our FDA-licensed cartridge technology, and Atlantic Horseshoe crab conservation efforts, please visit hsc.criver.com