Tradeoffs are a constant source of challenge in any research lab. To get faster results, you will probably need to use more resources (people, money, supplies). The powerful lasers used to do live cell imaging may well kill those cells in the process. Purifying DNA often leaves you to choose between purity and yield.
Working with biologics also involves a delicate balancing act. Producing compounds in biological models rather than by chemical synthesis offers many advantages, but it is not without certain challenges. One of those tradeoffs results from scaling up; the more plasmid that is produced, the greater probability of endotoxin contamination.
Recently, I had the opportunity to attend a fascinating symposium held at Promega featuring conservationist Steward Brand, where he described some of the projects developed by his foundation, Revive & Restore.
The organization’s mission is to apply emerging biotechnology techniques to endangered and extinct species with the intent to increase genetic diversity, provide disease resistance and facilitate adaptation to changing climates. Although the overall message of enhancing biodiversity through the application of new genetic technology was inspiring, the project that resonated most for me was related to the plight of horseshoe crabs.
Horseshoe crabs, often referred to as living fossils, include four extant species with origins dating back about 450 million years. Although they look like crabs, they belong to their own subphylum and are more closely related to spiders. When horseshoe crabs spawn, they leave their usual habitat on the ocean floor and migrate to shore in large numbers. As a result, they have been exploited for bait and fertilizer for decades.
Enter endotoxins, an indicator for bacterial contamination in biologicals, drugs and medical devices. U.S. Food & Drug Administration regulations dictate that finished products be tested for the presence of endotoxins. These pyrogenic compounds, found in the cell wall of Gram-negative bacteria, can cause fever and affect a wide range of biological activity, possibly leading to aseptic shock and death. The most common method for testing is the gel clot and Limulus Amebocyte Lysate (LAL) Test.
I first learned about the LAL test during graduate school, where it was presented as a ubiquitous and standard requirement for testing bacterial contamination in injectable drugs. I remember being fascinated that horseshoe crabs (Limulus sp.), contain a substance that could be used to detect endotoxins. Although the instructors mentioned the need to collect blood from horseshoe crabs in order to produce the test, the method or scale of this harvest wasn’t discussed, nor were the true costs of using this method of endotoxin testing.
The LAL test has served as a faster, more inexpensive endotoxin testing replacement for the rabbit pyrogens test for the past 35 years. Every year during mating season horseshoe crabs move to shallow water, where they are removed in huge numbers. (To get an idea of scale for the harvest and read a much more comprehensive investigation of the issue, check out this article in The Atlantic, which features an archive photo of Delaware Bay horseshoe crab harvest from 1928—for fertilizer, not pharmaceutical testing.)
After collection, the crabs end up in a lab where up to 30% of their blood is drained from a needle stuck in tissue around their heart. The LAL is extracted from the blood and can yield a product worth up to $15,000/quart. In order to avoid recollection, the crabs are returned to the ocean far from the shore where they were collected a few days before. Although it’s estimated that only 10-30% of these crabs die as a result of the process, there are indications that the horseshoe crab population and their ecosystems are impacted in other ways.
Researchers at the University of New Hampshire and Plymouth State University used accelerometers attached to recently bled female horseshoe crabs to test the hypothesis that harvesting for LAL was affecting their ability to spawn. While the research supported previous estimates with a death rate of 18%, females were found to be less likely to mate after being bled.
During his talk, Brand shared results from a study still in review that confirm the effect of over-harvesting Limulus on the survival of long distance migratory shorebirds. These birds synchronize their migration with horseshoe crab spawning, which provides a needed feast of eggs before the homestretch of their journey. Along with other ecosystem threats from climate change, the accelerated decline in the horseshoe crab population and dependency of migratory birds will likely to lead to a devastating ecological domino effect.
Fortunately, a synthetic alternative to LAL, recombinant factor C (rFC), has been available for nearly 20 years. Alas, there has been no significant shift by pharmaceutical companies away from the test based on horseshoe crab blood. rFC was patented and licensed to one company, Lonza, which Brand posited as one reason for the reluctance of drug companies to adopt its use.
Obviously, relying on one source for an essential testing reagent with no competition to temper cost is quite unattractive. But that argument has less bearing now that the patent is scheduled to expire in a few months, opening the door for additional manufacturers and creating an economic incentive for switching to the synthetic test.
Another reason may be that implementing a new test would require additional resources to validate the synthetic test for products that are already being tested with the LAL. Since the LAL has been specified in FDA guidance documents on endotoxin testing for decades, quality standards for existing products are based on the LAL, limiting momentum to change.
If both tests offered the same benefits, these arguments would make sense; however, research by one of the discoverers of rFC, Jeak Ling Ding of the National University of Singapore, shows that in many respects rFC is more efficacious than LAL. Since the raw material for the LAL test depends on an organism, there is seasonal variation in the components of the processed blood that must be taken into account. The reaction of the LAL also depends on a cascade of multiple compounds that can be affected by temperature, pH and proteins—leaving the test vulnerable to false positive results.
Although Eli Lilly is the only pharmaceutical company to date to use rFC in place of LAL, It seems the tide may be turning. According to Brand, others are interested in making the transition. It seems foolish not to, given the source for LAL shows signs of dwindling due to overexploitation. Perhaps pharmaceutical companies are beginning to see the value of a “slower/better” philosophy (the cornerstone of the Long Now Foundation, another brainchild of Brand’s), rather than “faster/cheaper.” I have certainly gained a new perspective on endotoxin testing and a deep appreciation for the work of Brand and his foundation.
Does your organization use the LAL test? What is preventing you from switching to the synthetic alternative? Let us know!
By clicking “Accept All”, you consent to the use of ALL the cookies. However you may visit Cookie Settings to provide a controlled consent.
If you are located in the EEA, the United Kingdom, or Switzerland, you can change your settings at any time by clicking Manage Cookie Consent in the footer of our website.
Necessary cookies are absolutely essential for the website to function properly. These cookies ensure basic functionalities and security features of the website, anonymously.
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Analytics".
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Functional".
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Other.
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Advertisement".
This cookie is set by GDPR Cookie Consent plugin. The cookies is used to store the user consent for the cookies in the category "Necessary".
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Performance".
6 months 2 days
This cookie is set by the provider Media.net. This cookie is used to check the status whether the user has accepted the cookie consent box. It also helps in not showing the cookie consent box upon re-entry to the website.
This cookie is used to store the language preferences of a user to serve up content in that stored language the next time user visit the website.
Analytical cookies are used to understand how visitors interact with the website. These cookies help provide information on metrics the number of visitors, bounce rate, traffic source, etc.
This cookie is associated with Sitecore content and personalization. This cookie is used to identify the repeat visit from a single user. Sitecore will send a persistent session cookie to the web client.
This domain of this cookie is owned by Vimeo. This cookie is used by vimeo to collect tracking information. It sets a unique ID to embed videos to the website.
1 month 18 hours 24 minutes
This cookie is used to calculate unique devices accessing the website.
This cookie is installed by Google Analytics. The cookie is used to calculate visitor, session, campaign data and keep track of site usage for the site's analytics report. The cookies store information anonymously and assign a randomly generated number to identify unique visitors.
This cookie is installed by Google Analytics. The cookie is used to store information of how visitors use a website and helps in creating an analytics report of how the website is doing. The data collected including the number visitors, the source where they have come from, and the pages visted in an anonymous form.
Advertisement cookies are used to provide visitors with relevant ads and marketing campaigns. These cookies track visitors across websites and collect information to provide customized ads.
1 year 24 days
Used by Google DoubleClick and stores information about how the user uses the website and any other advertisement before visiting the website. This is used to present users with ads that are relevant to them according to the user profile.
This cookie is set by doubleclick.net. The purpose of the cookie is to determine if the user's browser supports cookies.
5 months 27 days
This cookie is set by Youtube. Used to track the information of the embedded YouTube videos on a website.
Performance cookies are used to understand and analyze the key performance indexes of the website which helps in delivering a better user experience for the visitors.
This cookies is set by Youtube and is used to track the views of embedded videos.
This is a pattern type cookie set by Google Analytics, where the pattern element on the name contains the unique identity number of the account or website it relates to. It appears to be a variation of the _gat cookie which is used to limit the amount of data recorded by Google on high traffic volume websites.