Course:CONS200/2023/Horseshoe Crabs and the Biomedical Applications of their Blood

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Introduction

Horseshoe crabs are aquatic arthropods of the order Xiphosura. In spite of their name, they are not crabs, but actually arachnid arthropods, making them more related to spiders, scorpions, amblypygids, and other land-dwelling creatures than crabs[1]. Often referred to as "living fossils", horseshoe crabs have existed for around 455 million years, predating even dinosaurs[2]. This article focuses mainly on the American species Limulus Polyphemus, however, there are three other species of horseshoe crabs found in Asia: Tachypleus tridentatus (Japanese tri-spine horseshoe crab), Tachypleus gigas (coastal horseshoe crab), and the Carcinoscorpius rotundicauda (mangrove horseshoe crab)[1]. L. Polyphemus lives along the Atlantic and Gulf coasts, spanning from Maine to Mexico. Large groups of horseshoe crabs nest on beaches along Delaware, Maryland, and New Jersey[2].

Humans interact with horseshoe crabs for a variety of purposes. They are harvested for human consumption (mainly in Asia), as bait for the fishing industry, and most importantly, for their blood. Horseshoe crab blood is used to produce a substance called LAL, which is widely used in the global biopharmaceutical industry to test medication for endotoxins. Horseshoe crab populations have been declining as the result of overharvesting, raising ethical and ecological concerns[3]. Government and community efforts have been made to address this issue, as well as conservation efforts on the part of LAL manufacturing companies. However, further conservation efforts are needed in order to guarantee the long-term security of horseshoe crabs as a species. This article examines two proposed solutions to the issue: refinement of the harvesting and blood extraction, such as by ranching horseshoe crabs in an ideal environment; and the adoption of a synthetic alternative to LAL.

Limulus polyphemus (Atlantic Horseshoe Crab)

Implications for Health and Biodiversity

Properties and Application of Horseshoe Crab Blood

Horseshoe crabs have an innate immunity to pathogens, rather than an adaptive immune system. Their immune response relies on a type of mobile cell called granular amebocytes, which constitute 99% of a horseshoe crab's hemocytes (blood cells)[4]. This cell has the unique capacity to trigger coagulation of the blood when it encounters an endotoxin or 1,3ß-D- glucan, a component of the cell walls of fungi and bacteria[5]. The resulting clot immobilizes the pathogen, and is the basis for a form of endotoxin testing called the Limulus amebocyte lysate (LAL) test. LAL is produced by centrifuging and processing horseshoe crab blood, after which the solution is mixed with a pharmaceutical sample to test for endotoxins. If endotoxins are present, they will usually be detected as a gel clot[6].

The LAL test is used for all injectable medications and implantable medical devices, as mandated by the FDA[7]. Endotoxin testing is necessary to ensure human health and safety, as endotoxins can cause serious fever, septic shock, and death if injected[8]. Thus every injectable/implantable pharmaceutical product imported or exported by the US undergoes LAL testing, making it the dominant endotoxin detection method in North America and Europe[9].

Harvesting Horseshoe Crab Blood

Every year, roughly 500,000 horseshoe crabs are harvested along the Eastern

Horseshoe crabs are bled at the Charles River Laboratory in Charleston, South Carolina, US

coast of the United States for biomedical use[10][6]. They are harvested from shallow waters using dragging trawls, then are stacked on boats before being packed into plastic containers for transport[11]. Horseshoe crabs then spend up to 24 hours in transit from the seashore to a lab, where their blood will be extracted. A hypodermic needle is used to extract 50-400 millilitres of blood per crab, after which most crabs are immediately returned to their habitat, although around 13% will be sold as bait for eel and whelk fishing[6][11].

Associated Risks

The mortality rate for horseshoe crabs post-bleeding ranges from 15-30%, and likely falls on the higher side, accounting for their use as bait[10][11]. Surviving crabs recover their blood volume within 3-30 days of their release, while the reconstitution of amebocytes takes roughly 4 months[10]. Aside from mortality, there are several non-fatal health risks associated with the capture, transport, and bleeding of horseshoe crabs, including decreased mobility, compromised blood quality, and potential effects on spawning behaviour[12][10]. Once captured, crabs are stacked on a boat and in plastic containers, causing some to be crushed or impaled by other crabs[11]. Their removal from water poses an additional threat to their health– removal from water for a mere 5 minutes can cause severe hypoxia and metabolic acidosis[13]. Temperature regulation during transportation and holding is also poor, and exposure to higher temperature can cause a decline in horseshoe crabs' health and blood quality[14]. One study comparing bleeding methods found that 'non-stressed' crabs– those removed from the water, bled on the spot, then instantly returned– had a 6% mortality rate and thus fared much better than 'stressed' crabs– those subjected to the stressors of capture, transport and holding[12].

Ethical Concerns

As demonstrated by mortality rates and behavioural changes, the capturing and bleeding of horseshoe crabs causes significant stress to individual crabs and is thus not an animal harm-free method of endotoxin testing. However, endotoxin testing is necessary by legal and ethical standards, and because endotoxins can cause severe health complications and death in patients, consideration for the harm to horseshoe crabs has been minimal.

While the IUCN Red List assesses horseshoe crabs as a vulnerable species rather than endangered, it notes that their populations are fragmented, and populations in key areas such as the Gulf of Maine region and the Mid-Atlantic region qualify as endangered[3].

Due to the lack of recognition of horseshoe crabs as endangered species [15], and, up until recently, no viable alternatives to LAL, a majority of scientists fail to assess the use of horseshoe crabs by the 3R’s standard[9]. Coined by Russel and Birch[16], the 3Rs encompasses the (1) Refinement of research by ensuring the welfare of animal participants, (2) Reducing animal-based testing, and eventually, (3) achieving viable Replacements. The 3Rs framework is widely implemented and agreed upon as a means to uphold moral value while maintaining quality science[9]. However, the guideline is predominantly applied to preserve species listed within formal legislations regarding animal use[9], and in many countries, horseshoe crabs are overlooked[15].

Of the four species remaining today, the tri-spine horseshoe crab (Tachypleus tridentatus) native to the coasts of Asia, and the American horseshoe crab (Limulus polyphemus) inhabiting the Gulf of Mexico, are receiving slight attention, as they are deemed ‘moderately depleted’ by the International Union for Conservation of Nature (ICUN), though their numbers are rapidly declining[15].

Once LAL extraction is completed, horseshoe crabs are returned to their habitats [15]. However, as noted, studies highlight that their reintegration can be met with abnormal behavioural changes[10] and decreased survival rates [17]. Other studies uncover a short-term increase in mortality rates depending on the amount of blood extracted [17]. The results portray an increased mortality rate by 29.4% when 40% of hemolymph volume is harvested [17]. In other words, of the 500,000 L. polyphemus collected, approximately 147,000 individuals are not expected to survive upon returning to their habitats [17]. The findings from these studies suggest blood extraction is likely to decrease the short-term survivability, and healthy spawning and foraging behaviours of horseshoe crabs [10], which conflicts with the 3Rs principle of retaining animal wellbeing during scientific expeditions[16].

Dwindling horseshoe crab populations and decreasing spawning rates have also raised concern due to their role in the ecosystem, as an array of marine and avian species rely on horseshoe crab eggs for sustenance[18]. The overharvesting of crabs thus has rippling impacts on the surrounding ecosystem.Their ecological role as primary food sources should be emphasized in order to mobilize pharmaceutical companies to work toward viable replacements for their LAL cells.

Red Knot feeding on the eggs of horseshoe crabs

Contributors to Biodiversity and Ecosystem Functions

Horseshoe crabs are a crucial component of marine Atlantic ecosystems. Horseshoe fulfill the role of bioturbators, hosts for various epibionts (organisms that live on the surface of other living organisms, controllers of benthic invertebrate populations, and a food source for other animals[18].

As bioturbators, Horseshoe crabs rework the soil and sediment through burrowing, ingestion, and defecation; providing an essential service for sea floor organisms by distributing organic matter. Their crustaceous shells also provide a habitat for epibionts, such as barnacles[18]. Additionally, Horseshoe crabs control seafloor benthic invertebrate populations, especially surf clams and blue mussels, through their vacuum feeding practices. Many animals, including turtles, crabs, pufferfish, and migratory birds rely on horseshoe crabs and their eggs as an essential part of their diet[18].

Horseshoe crabs are considered a keystone or indicator species due to their heavily intertwined role in the ecosystem. This means that their condition and health is deeply linked to the state of the surrounding ecosystem. One species for which horseshoe crabs are particularly important is the threatened red knot shorebird. Crabs spawn in Delaware Bay from May to June, corresponding with migration of shorebirds including the red knot. The shorebird relies heavily on horseshoe crab eggs as sustenance for their migration. Springer, US, states “Survival and successful reproduction of the many migratory shorebirds that stop to feed at Delaware Bay are strongly linked to horseshoe crab reproduction” [18] The red knot population requires 15.4 billion eggs to sustain their migration which equates to the production of about 170,000 female Horseshoe crabs[18]. With a steady decline of Horseshoe crab populations, these shorebirds struggle for food on their migration, threatening their survival.

The recent decline in horseshoe crab populations creates increasingly negative impacts on the environment to which they are crucial, and this threat promises to increase with the expansion of the pharmaceutical market. Current harvesting practices considered, the horseshoe crab is likely to decline further, and without the horseshoe crab's presence in their ecosystems, it is likely these ecosystems will collapse[18].

Conserving the Horseshoe Crab

Government Action

With the recognition of T. tridentatus as 'endangered' and L. polyphemus as 'vulnerable', in the International Union for Conservation of Nature (IUCN) Red List of Threatened species™ governments began intensifying their global conservation efforts [19]. The IUCN Species Survival Commission (SSC) Horseshoe Crab Scientific Specialist Group (SSG) conducts workshops aimed at raising global awareness on the critical state of Limulidae while formulating management plans [19]. Upon the completion of a workshop in Guangxi, China, participants representing 14 countries endorsed the Beibu Gulf Declaration on Global Horseshoe Crab Conservation, which calls for the sustainable use of horseshoe crabs, restoration of remaining populations and expansion of critical habitats[19].

Community Responses

Beached Limulidae attempting to right itself

Communities across the globe have begun addressing Limulidae population scarcity through the implementation of educational programs that work to increase the public’s awareness and engagement towards conservation [20]. The Ecological Research & Development Group (ERDG), based in the US, introduced a ‘Just Flip em’!’ campaign in the 1990s, encouraging communities to flip over any horseshoe crabs found on their backside [21]. During spawning, horseshoe crabs are often flipped over due to waves along the the shoreline [22]. Approximately 10-15% of horseshoe crabs are unable to help themselves out of this predicament, leading to death by predation or desiccation. [22]. Thus, in promoting this simple act, a significant number of horseshoe crabs can be saved and the public can develop a connection with these unique animals [21]. These human-animal bonds help to inspire continued learning on conservation efforts and sharing knowledge with fellow community members [21]

In addition to campaigns, institutions such as the City University of Hong Kong (CityU), China, have introduced courses based on T. tridentatus (Chinese horseshoe crab) preservation [20]. Programs conducted by the Ocean Park Conservation Foundation Hong Kong (OPCFHK) and CityU focus on fostering a relationship between students and T. tridentatus and decreasing ingrained apathy towards conservation by providing them with the opportunity to raise laboratory-cultured animals [20]. With the development of a human connection to T. tridentatus and relatively successful species survival rates of 76.2%, students reported gains in knowledge of the ecology of T. tridentatus, and an increase in self-efficacy regarding protection efforts [20]. Thus, further implementation of institutional education programs centred on the needs of Limulidae could increase public responsibility and positive attitudes towards vulnerable species conservation [20].

Manufacturers' Efforts

Facing a decline in horseshoe crab populations, many harvesting companies have made efforts to decrease catch rates in order to ensure the long-term survival of the species[23]. For example, LAL-manufacturing company Lonza has stopped selling horseshoe crabs for bait after bleeding them, and donates funds annually to education programs and coastline community conservation efforts[24]. In China, invasive fishing mechanisms, such as trawling, involve the destruction of habitats through dragging heavy nets along the ocean floor [23]. The aggressive fishing techniques, along with pollution caused a decrease in T. tridentatus populations by > 90% [23]. In an effort to preserve remaining Limulidae populations, a workshop involving 40 scientists from Taiwan, mainland China and Hong Kong, banded together to create a regional 'wise use' program known as the Horseshoe Crab Conservation Consortium [23]. This program aims to reduce illegal use and trade of Limulidae, maintain consistent monitoring of their conservation status, and ensuring fisheries adhere to sustainable catch rates, allowing an adequate amount of crabs left to recuperate after harvest [23].

Safer Harvest and Extraction Techniques

As evidenced by Hurton et. al, the transport and storage of horseshoe crabs may be a stronger cause of fatality than the bleeding process itself. They demonstrate a remarkably low rate of mortality (6%) among non-stressed crabs who only subjected to bleeding, and not transport and holding[12]. Thus bleeding crabs on the spot rather than in a lab, or improving the conditions under which they are transported and stored poses a potential opportunity for refinement. In the latter option, biopharmaceutical companies would need to closely manage the practices of the fishermen under contract[9]. However, Krisfalusi-Gannon et. al highlights the potential of this method, stating that it could decrease mortality rates by one-half[11].

Another proposed technique is the aquaculture and maintenance of captive horseshoe crab populations. This system has the benefit of protecting vulnerable horseshoe crabs– including juveniles, and those recently bled– from stressors including predation and human disturbance; as well as removing the steps of harvest and transport from the bleeding process. It would also allow for the extraction of smaller amounts of blood on a more frequent basis, and there has been some research to show that aquaculture under the proper conditions can increase horseshoe crab health[9]. However, some scientists note that we do not currently understand the conditions of the optimal horseshoe crab environment enough to ensure the welfare of the crabs through aquaculture[9]. Additionally, many studies note that crabs suffer from chronic mortality within 6 months of captivity, potentially caused by a poor understanding of their diet[9].

A Synthetic Alternative to Horseshoe Crab Blood

In 1997, researchers at the University of Singapore cloned a single protein belonging to the horseshoe crab to create recombinant Factor C (rFC), a reagent capable of detecting endotoxins[6]. When it comes into contact with an endotoxin, rFC undergoes a chemical reaction resulting in a fluorescent compound, allowing a sample's endotoxin concentrations to be determined by level of fluorescence[6]. A study conducted by Maloney et al. confirms the efficacy and viability of rFC as a replacement for LAL using a synthesis of 10 independent studies conducted from 2009 to 2017[6]. These studies confirm that rFC is comparable to LAL in its sensitivity to endotoxins. Additionally, rFC tests demonstrated a higher rate of reliability that LAL based tests[25]. Unlike LAL, rFC does not contain glucan-sensitive factor G, a protein which can react with non-pathogenic compounds to produce false positives when testing for endotoxins. The largest-scale study to date on rFC efficacy consisted of similar, independent experiments conducted by pharmaceutical manufacturers including AstraZeneca, MedImmune, and Eli Lilly, which compared 37 LAL and rFC reagent and supplier combinations. This study concluded that rFC tests can detect endotoxins with a high limit of detection, and is comparable to, if not superior to LAL-based testing methods.[26]

Barriers to the Adoption of rFC

rFC reagents have been commercially available since 2003, and were recognized in 2012 by the FDA and the European health ministry as an acceptable alternative to LAL[6]. However, its adoption by the pharmaceutical industry has been slow. rFC is currently classified as an 'alternative test' in medical compendia such as the US Pharmacopoeia, upon which regulatory bodies including the FDA are based[9]. This means that the rFC method requires additional validation in order to meet regulatory requirements, which demands more time and money on the part of pharmaceutical testing companies. In 2020, the European Pharmacopoeia (Ph.Eur) designated rFC-based endotoxin testing as a compendial method, rather than an alternative method, eliminating the extra steps required for validation. However, due to the global scale of pharmaceutical markets, it is necessary to implement this designation across Pharmacopoeias in order for rFC testing to become a globally accepted compendial method[9]. Additionally, employees of the pharmaceutical sector have expressed trepidation regarding the status of rFC as a novel, less tested endotoxin testing method[9]. While studies on the effectiveness of rFC have demonstrated positive results, this aversion of risk reflects the weight of the decision– millions of patients' safety depends upon effective, reliable endotoxin testing. Thus some employees of the pharmaceutical sector would prefer to see more data on rFC safety and effectiveness. However, others argue that this data is already available, and therefore the question of confidence in rFC-based endotoxin tests is a matter of educating pharmaceutical personnel, rather than conducting more research.[9] The slow uptake of rFC by the pharmaceutical industry can thus be accounted for by the extra validation it requires, and hesitancy or aversion to potential risk among pharmaceutical personnel.

Conclusion

Horseshoe crabs, a unique species nearly unchanged by evolution for 445 million years, have evolved as a key component of their respective niche. A key factor in this domination is their unique immune system, which unlike ours, attacks threats with a blanket response. This blanket response is led by their granular amebocytes, which detect intruders and trigger a reaction which smothers them via the coagulation of blood, preventing their growth and spread. The very strength that led to their domination however, has also made them vulnerable to exploitation by humans. The unique ability of these amebocytes to coagulate quickly upon contact with endotoxins has made them extremely useful in ensuring that intravenous medical equipment and medicine isn't contaminated. The collection of horseshoe crab blood is meant to be non-lethal, however, the significant amount of blood taken and highly stressful conditions lead to a high mortality rate and undeniable adverse effects on survivors. This exploitation has reverberating effects on their ecosystem and the species that depend on them for survival, in addition to threatening the long term security of horseshoe crabs themselves. Preventive measures must be taken in the near future to avoid these consequences from escalating. Thankfully, the existence of a synthetic alternative provides pharmaceutical companies with a substitute option which proves just as effective without the environmental impacts of the LAL method. The adoption of RFC combined with additional conservation efforts such as reducing the allowable uptake for bait will allow for a steady increase in horseshoe crab populations and return to normalcy for the ecosystems that depend on them.

For this reason, we recommend the adoption of recombinant Factor C (rFC) to reduce the demand for horseshoe crab harvesting. It faces two main barriers: a lack of harmonization across pharmacopoeias, and hesitancy among biopharmaceutical manufacturers. The listing of RFC as an alternative testing method in most pharmacopoeias means that pharmaceutical manufacturers must overcome additional barriers to use it. These additional barriers are compounded by the high risk nature of endotoxin testing, as false negative tests could expose endotoxins directly into the bloodstreams of millions of people. The presence of this risk makes many pharmaceutical companies hesitant to switch away from a method which has proven to be so effective for so long. Despite this, numerous studies have proven the efficacy of rFC. Thus, we recommend governments initiate the adoption of rFC by listing it as a primary testing method in pharmacopoeias, and by implementing educational programs for biopharmaceutical personnel concerning the benefits of rFC in order to ease any doubts regarding its effectiveness. Additionally, we recommend the protection of spawning areas for horseshoe crabs as Category V protected areas in order to promote their recovery. Finally, the adoption of rFC may provide a window for the bait industry to rapidly increase their use of horseshoe crabs, thus legislation must be imposed to maintain or decrease the current number of crabs allocated to the fishing industry.

References

  1. ↑ 1.0 1.1 Cuvelier, Daphne (04/04/2004). "World Register of Marine Species". WoRMS taxon details. Check date values in: |date= (help)
  2. ↑ 2.0 2.1 "Horseshoe Crab". National Wildlife Federation. |first= missing |last= (help)
  3. ↑ 3.0 3.1 "IUCN Red List of Threatened Species". International Union for Conservation of Nature. February 2016. Retrieved April 10 2023. |first= missing |last= (help); Check date values in: |access-date= (help)
  4. ↑ Medzhitov, Ruslan; Janeway, Charles (2000). "Innate immune recognition: mechanisms and pathways". Immunological Reviews. 173: 89–97.
  5. ↑ Isakova, Victoria; Armstrong, Peter (October 2003). "Imprisonment in a Death-Row Cell: The Fates of Microbes Entrapped in the Limulus Blood Clot". The Biological Bulletin. 205: 203–204 – via JSTOR.
  6. ↑ 6.0 6.1 6.2 6.3 6.4 6.5 6.6 Maloney, Tom; Simmons, Naira (2018). "Saving the horseshoe crab: A Synthetic Alternative to Horseshoe Crab Blood for Endotoxin Detection". PLoS Biology. 16 – via PubMed Central.
  7. ↑ "Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers". U.S. Food and Drug Administration. June 2012 (updated March 2018). Check date values in: |date= (help)
  8. ↑ Nakamura, Takanori; Morita, Takashi (1986). "Lipopolysaccharide-sensitive serine-protease zymogen (factor C) found in Limulus hemocytes". European Journal of Biochemistry. 154: 511–521 – via EBSCO.
  9. ↑ 9.00 9.01 9.02 9.03 9.04 9.05 9.06 9.07 9.08 9.09 9.10 9.11 Gorman, Richard (2020). "Atlantic Horseshoe Crabs and Endotoxin Testing: Perspectives on Alternatives, Sustainable Methods, and the 3Rs (Replacement, Reduction, and Refinement)". Frontiers in Marine Science. 7 – via Frontiers.
  10. ↑ 10.0 10.1 10.2 10.3 10.4 10.5 Anderson, Rebecca; Watson III, Winsor; Chabot, Christopher (2013). "Sub-lethal behavioral and physiological effects of the biomedical bleeding process on the american horseshoe crab, limulus polyphemus". The Biological Bulletin (Lancaster). 225: 137–151 – via The University of Chicago Press Journals.
  11. ↑ 11.0 11.1 11.2 11.3 11.4 Krisfalusi-Gannon, Jordan; Ali, Waleed (2018). "The Role of Horseshoe Crabs in the Biomedical Industry and Recent Trends Impacting Species Sustainability". Frontiers in Marine Science. 5.
  12. ↑ 12.0 12.1 12.2 Hurton, Lenka; Berkson, Jim (2006). "Potential Causes of Mortality for Horseshoe Crabs (Limulus Polyphemus) during the Biomedical Bleeding Process". Fishery Bulletin (Washington D.C.). 104: 293–298.
  13. ↑ Allender, Matthew; Milam, Jennifer (2010). "The effects of short- and long-term hypoxia on hemolymph gas values in the American horseshoe crab (Limulus polyphemus) using a point-of-care analyzer". Journal of Zoo and Wildlife Medicine. 41: 193–200.
  14. ↑ Coates, Christopher; Bradford, Emma (2012). "Effect of temperature on biochemical and cellular properties of captive Limulus polyphemus". Aquaculture. 334-337: 30–38.
  15. ↑ 15.0 15.1 15.2 15.3 Gallagher, Katherine (October 2022). "Are Horseshoe Crabs Endangered? Conservation Status and Threats". Treehugger.
  16. ↑ 16.0 16.1 Russel, William; Burch, Rex (1959). The principles of humane experimental technique. Methuen.
  17. ↑ 17.0 17.1 17.2 17.3 Hurton, Lenka; Berkson, Jim; Smith, Stephen. "The Effect of Hemolymph Extraction Volume and Handling Stress on Horseshoe Crab Mortality". Biology and Conservation of Horseshoe Crabs: 331–346 – via Springer US.
  18. ↑ 18.0 18.1 18.2 18.3 18.4 18.5 18.6 Bottom, Mark (2009). "The Ecological Importance of Horseshoe Crabs in Estuarine and Coastal Communities: A Review and Speculative Summary". Springer US: 45–63.
  19. ↑ 19.0 19.1 19.2 Tanecredi, John T.; Bottom, Mark L.; Shin, Paul K. S.; Iwasaki, Yumiko; Cheung, Siu Gin; Kwan, Kit Yue; Mattei, Jennifer H. (2022). Conservation of horseshoe crabs species globally. Springer International Publishing.
  20. ↑ 20.0 20.1 20.2 20.3 20.4 Kwan, Billy K. Y.; Cheung, Joe H. Y.; Law, Anniqa C. K.; Cheung, S. G.; Shin, Paul K. S. "Conservation education program for threatened asian horseshoe crabs: A step towards reducing community apathy to environmental conservation". Journal For Nature Conservation. 35: 53–65 – via Elsevier.
  21. ↑ 21.0 21.1 21.2 Ecological Research and Development Group. "Just flip em'!". The Horseshoe Crab.
  22. ↑ 22.0 22.1 Kreamer, Gary; Kreamer, Sharon W. "Green eggs & sand, team limulus, and more: Educating for horseshoe crab conservation in the united states". Changing global perspectives on horseshoe crab biology, conservation and management : 557–574 – via Springer International Publishing. no-break space character in |journal= at position 84 (help)
  23. ↑ 23.0 23.1 23.2 23.3 23.4 Lin, Wuying; Chen, Chang-po; Shin, Paul. "Collaborative conservation for horseshoe crabs". Oryx. 48: 482–483 – via Cambridge Core.
  24. ↑ "Horseshoe Crab Conservation". Lonza. Retrieved April 2023. |first= missing |last= (help); Check date values in: |access-date= (help)
  25. ↑ Ding, Jeak; Ho, Bow. "A new era in pyrogen testing". Trends in Biotechnology. 19: 277–281. doi:10.1016/S0167-7799(01)01694-8 – via CellPress.
  26. ↑ Bolden, Jay; Knight, Michael (July 2017). "Results of a harmonized endotoxin recovery study protocol evaluation by 14 BioPhorum Operations Group (BPOG) member companies". Biologicals. 48: 74–81 – via Elsevier Science Direct.


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