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Strange ally in the war against sepsis: 50 years ago this year, scientists first connected an ancient sea creature to testing for life-threatening bacteria.

As summer winds down, many of us recall memories of beach trips past. Perhaps some of you might even remember the first such trip where you saw the distinctive form of a horseshoe crab (photo). The species dates back to the Ordovician period, some 450 million years ago. Few people outside of the pharmaceutical industry realize how many human lives that these crabs may have saved.

Marine biologists had studied the unique properties of the creature's blood and immune system, and 50 years ago this year, the first academic paper was published describing a method in which horseshoe crab blood might be used to detect pyrogenic bacteria.

This discovery eventually led to the development of systems for detecting endotoxins, outer membranes of gram-negative bacterial cell walls. When present in high concentrations in pharmaceutical products or processing equipment, endotoxins can lead to toxic shock, sepsis and death, and diseases such as meningitis. Gram negative sepsis is said to affect 300,000 people in the U.S. each year. The company, EndoSafe was set up to market technology based on limulus amebocyte lysate (LAL), a key ingredient in the horseshoe crab's blood, which reacts in the presence of gram-negative bacteria.

The first commercial endotoxin detection systems based on LAL were approved by FDA and commercialized in 1989. Since then, EndoSafe has become part of Charles River Laboratories, and, eight years ago, the company developed rapid LAL-based endotoxin systems for monitoring product and processes

John Dubczak, general manager of Charles River Laboratories' Endotoxin and Microbial Detection division, recently shared trends in the rapid microbiological testing market, and the history of LAL-based systems.

Contract Pharma: Over the past few years, numerous FDA Warning Letters and 483's have pointed to insufficient endotoxin monitoring practices. Have there been any changes in regulation or enforcement, lately?

John Dubczak: There haven't been any changes. However, FDA's Process Analytical Technology (PAT) guidelines and its revised Process Validation guidance have been very relevant to rapid microbial methods, including Endosafe. These guidances reflect an emphasis on process understanding and control and knowledge of critical quality attributes that will affect the quality of finished product.

About two years ago, FDA updated its original 1987 guidelines covering the use of LAL testing for in-product release in an LAL-testing Q&A (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatorylnformation/Guidances/ UCM310098.pdf).

CP: Why are we seeing so many more examples of companies failing to prevent endotoxin contamination in their facilities and their product?

JD: Biopharmaceutical manufacturing is becoming much more complex, especially for targeted therapies and vaccines. These operations are very sophisticated and involve the use of living cells which are vulnerable to contamination.

CP: Has FDA's position on rapid microbial monitoring been positive, and do you see that having an impact on its adoption in pharma?

JD: Many technologies have been developed to rapidly identify contamination, and FDA has reacted favorably. Testing is expected at the process level, and a lot of organizations are working on that. Regulators have been cautious. LAL testing was introduced in the 1970's. FDA recognized its potential, published notices in the Federal Register, and allowed companies to use it on a limited basis. But it still took 15 years for the agency to approve the technology and issue its 1987 guidance.

CP: When was your rapid test method introduced?

JD: FDA approved it in July of 2006. We simply took reagents that the FDA approved in 1993 and applied it to disposable plastic cartridge (Figure). Each cartridge features four reaction wells--sample is analyzed in two of the wells, and the two other wells are used as positive product controls to rule out the possibility of false negatives. The rapid system has made testing very easy, generating quantitative data in less than 15 minutes, compared to the four to eight (or more) hours required for the old method.

CP: How did the whole technology come about?

JD: It's an example of how basic research and basic science play such a key role in technology innovation. In the 1950's, Johns Hopkins marine biologist Dr. Frederic Bang, studied horseshoe crab blood clotting in the presence of gram-negative bacteria. He published papers on this observation and these findings sat in the literature for 10 years.

In the mid 1960's, fellow Johns Hopkins scientist, hematologist Jack Levin was researching potential clinical applications involving the detection of gram-negative bacteria, responsible for septic shock, in the blood. He did a literature search and found Dr. Bang's paper. The two collaborated to develop a clinical test.

Then Dr. James Cooper (also at Johns Hopkins) used the reagent of Levin and Band and applied it to the testing of radiopharmaceuticals used to diagnose cancer. These materials have very short half lives and would deteriorate during the five hours required for traditional pyrogenic testing.

Cooper's research demonstrated that the LAL test was more sensitive than the rabbit pyrogenic test. He applied the technique to PET agents and published a paper on this work in 1971. Both FDA and NIH took note, and subsequent papers explored the use of LAL in other applications.

Dr. Cooper founded Endosafe in 1987. Two years later, the technology was licensed by FDA, allowing Endosafe to produce laboratory reagents for sale in the U.S. In 1991, the company introduced kinetic turbidometric reagents that are licensed for both kinetic turbidometric and gel clot methods, and chromogenic reagents two years later. Charles River acquired the company in 1994. The rapid method came to market in 2006.

CP: There are a number of rapid microbial monitoring systems out there now, and some of them are applicable for endotoxin testing. Could some of these alternative systems displace LAL methods in some applications?

JD: Other methods are available, including one based on recombinant human factor C, but FDA approval may take extensive validation, and time.

CP: Could tightening supplies of LAL, and a dwindling crab population, be a factor in the future?

JD: It's not a factor at all, at least in South Carolina where the crab population is not endangered. Other states allow the crabs to be used as bait, but South Carolina protects the species, and hasn't allowed them to be "landed" since 1992. Dr. Cooper has worked with the state to actively protect the crabs, and South Carolina's crab population is actually increasing. In addition, the rapid LAL testing method uses one-twentieth the amount of LAL that the original test did.

Agnes Shanley

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Author:Shanley, Agnes
Publication:Contract Pharma
Date:Sep 1, 2014
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