Stars of the Pharma Sea

On new moon and full moon nights, as the waves boil and break on the coastline of Odisha and West Bengal, an ancient sea creature scuttles ashore to spawn, often in mating pairs. Under its carapace are secrets to fundamental biological processes, and blue blood that clots upon encountering certain microbes, making it the universal gold standard for identifying bacterial contaminants in drugs. For every vaccine and injectable drug we have ever taken, we have the American horseshoe crab, Limulus polyphemus, to thank. Nature’s best pharmaceutical chemist, the horseshoe crab, is a marine arthropod that is thought to have originated 450 million years ago, and survived the Permian extinction. It has fought long and hard for its place in the world, and learned to survive in the harshest circumstances. Horseshoe crabs can go up to a year without food, have no major predators and survey the ocean for mates with 10 eyes. In fact, a study of their compound eyes by Keffer Hartline in the early 20th century led to a Nobel Prize-winning research on the neurophysiology of vision. Evolution, however, played a cruel trick on the organism when humans discovered 60 years ago that its primitive immune system can detect and kill invading bacteria. First introduced commercially in the 1970s, the limulus amebocyte lysate (LAL) test, derived from horseshoe crab blood cells, has since become de rigueur for detecting endotoxins—pyrogenic components found in the outer membrane of bacteria such as E. coli, pseudomonas and salmonella—in pharmaceuticals and medical devices before final release.

Over 600,000 horseshoe crabs are bled every year in American facilities by a handful of companies that dominate the LAL endotoxin testing market, and released back into the sea. Although the harvest is regulated by the Atlantic States Marine Fisheries Commission (ASMFC), an estimated 10 per cent of the creatures succumb to the trauma. The keystone species is now under the spotlight as the race to find a vaccine for Covid-19 heats up, sending pharma majors across the world into a frenzy of development and testing. The International Union for Conservation of Nature (IUCN) had, in 2016, predicted declines of at least 30 per cent of the American horseshoe crab population over the next 40 years. This decline is now expected to be hastened by the increasing global demand for vaccines, pharmaceuticals and medical devices requiring LAL testing workflows. Among the alternatives to the LAL test is TAL (tachypleus amebocyte lysate), extracted from the Asian Tachypleus tridentatus, produced mainly by China for the Asian market, and an animal-free assay based on a synthetic equivalent of factor C, a component of the horseshoe crab blood-clotting cascade that gets activated in response to endotoxins. Despite the globally recognised need to transition to sustainable synthetic replacements for LAL/TAL assays, adoption of the recombinant factor C (rFC) test has remained limited so far. Few companies that offer endotoxin testing solutions, including the Swiss Lonza group and the French bioMérieux, have skin in the rFC game. In fact, the American medical standards group US Pharmacopeia (USP) recently decided against giving rFC equal standing with horseshoe crab blood, citing the need for more validation.

Notwithstanding the inertia in endotoxin testing, Indian scientists say there has never been a better time to pursue homegrown alternatives to LAL. Home to two of the four extant species of the horseshoe crab, the Carcinoscorpius rotundicauda, which inhabits mangroves, and the coastal Tachypleus gigas, India has listed both under Schedule IV of its Wildlife Protection Act, 1972. Scientists have, over the years, explored sustainable ways of extracting blood from the crabs, worked with fishermen and local communities to rescue and release strays back into the sea and to conserve their habitats, and proposed synthetic alternatives. “Unlike in China, where horseshoe crab populations are threatened by over-harvesting, the only threat to these crabs in India so far has been due to deterioration of habitat and accidental trapping in fishing nets. The crab is not edible. In northern Odisha, some tribes boil it in mustard oil until it turns blue and bottle and sell it as medicine for rheumatism, but this is not a large-scale activity,” says Siddhartha Pati, a researcher at the Institute of Tropical Biodiversity and Sustainable Development (Bio-D Tropika), University of Malaysia, Terengganu, and an IUCN specialist on horseshoe crabs in India. Pati works with the Association for Biodiversity Conservation and Research in Balasore, Odisha, to create awareness among locals in the region and has led a Wildlife Trust of India-funded project to rescue thousands of T. gigas specimens entangled in fishing nets long the sandy beaches of Balaramgadi. “The horseshoe crab species found in India are classified as ‘data deficient’ in the IUCN list—we are working on bumping them up to ‘vulnerable’ status. There hasn’t been a population survey yet, but we know that the coastal crabs are abundant, although cyclones, erosion and development activities are increasingly impacting their habitats. We find crabs with broken tails and morphological abnormalities that weren’t observed until a few years ago,” Pati says. A conservationist, Pati is not averse to sustainable lysate production from T. gigas crabs and biochemical research that could lead to crucial drug discoveries. The horseshoe crab produces a host of molecules that have been known to be biochemically valuable, as is evidenced by a study undertaken by the Queensland University of Technology pointing to a defence peptide, tachyplesin-I, as a promising anticancer drug lead. “There is much that is yet unknown about the horseshoe crab and only sustained interest from the scientific community can help underscore the importance of the species so it can be studied and conserved better. For instance, researchers at the Fakir Mohan University, Balasore, released over 15,000 artificially-fertilised and ranched crabs into the Bay of Bengal—thanks to a three-year Department of Biotechnology grant,” says Pati, who has co-authored research based on controlled experiments to improve cardiomyocyte regeneration using biocompounds found in minute quantities inside T. gigas eggs.

THE WORLD NEEDS 70 million endotoxin tests annually and the market for these is expected to cross the $1 billion mark by 2024. The LAL reagent market in India, according to an industry expert, is worth Rs 800 million, or $10.6 million, a year, but with the Indian pharma sector sensing opportunity in the Covid-19 pandemic, demand could shoot up in the near future. The third largest producer of pharmaceuticals in the world by volume, India is also one of the world’s top suppliers of measles, diphtheria, tetanus and tuberculosis vaccines.

“The opportunities for India are manifold—we not only have a sizeable well-conserved population of horseshoe crabs, we also have the ability to produce synthetic alternatives,” says Anil Chatterji, a retired scientist at the Indian Institute of Oceanography who holds a lapsed patent for producing lysate from Indian horseshoe crabs. Chatterji, who walked the length of the coast from the Sunderbans to Paradip to study horseshoe crabs in the mid-1980s on commission for a Mumbai-based pharma company, claims the lysate that his lab produced was as good as LAL, but the path to the market wasn’t straightforward. “The limited factor was not that T. gigas being smaller than the American crab, we would have to catch a lot more of them, it was that testing and validation on a large-scale required sizeable investments and at the time, productising the reagent wasn’t viable,” he says. “Now, the world is more open to LAL alternatives, especially synthetic ones.”

Chatterji co-authored a paper with SK Pal from the Indian Institute of Science Education and Research, Mohali, about a new-generation liquid crystal gel for rapid detection of endotoxins. “There have been advancements towards developing a reusable liquid crystal gel slide that can detect all sorts of bacteria, not just the gram-negative-type detectable by crab blood,” says Pal, who began working on the technology as part of his postdoctoral work at the University of Wisconsin (UW), Madison. In a paper published in Science in May 2011, researchers at UW-Madison reported that micrometre-sized liquid crystal droplets changed in response to minute quantities of endotoxins. The phenomenon is yet to be productised, however. “One of the problems is that stabilisation of liquid crystal droplets is difficult. When we try to restrict the flow of the liquid and turn it into thin films of gel that can be easily transported and marketed as a portable endotoxin detection kit, the sensitivity is affected. For use in pharma, we need to achieve nanogram/ml sensitivity and we are working towards this,” Pal says. “India has a number of qualified liquid crystal chemists, but not many companies that can handle the materials. As we speak, we are on the lookout for funding to productise the tech—hopefully, now with the resurgence in interest in synthetic alternatives to LAL, we may get an opportunity.”

“There is no dearth of government funding. The DBT funds basic research and then there are agencies like the Biotechnology Industry Research Assistance Council (BIRAC) that fund translational projects. But between the proof of concept and the market shelf, there is a valley of death,” says a senior DBT official. “Indian scientists could use a little hand-holding.” The problem with introducing a new reagent, says Anurag S Rathore, at the Centre of Excellence in Biopharmaceutical Technology, IIT Delhi, is the inevitably slow business development cycle. “Product adoption is primarily done by drug manufacturers who cannot just switch to a new test overnight. It is not about getting the technology to work, it is about gaining widespread acceptance over time and that takes decades of consistent research,” he says.

A representative of Lonza India says that its rFC test has not been used by any Indian pharma company to release a drug into the market. “Competitors have spoken up against promoting a monopoly and adopting new standards and even if we know this is the sustainable route, there have been hiccups,” he says. “We don’t see ourselves using alternatives to the LAL test unless they become widely accepted,” says Bala Reddy, head, biologics and biosimilars at Hetero Labs, a Hyderabad-based pharma company that is one of the five generic drugmakers to have inked temporary royalty-free licensing deals with Gilead Sciences Inc to produce Remdesivir, an antiviral drug approved by the US Food and Drug Administration for emergency use to treat Covid-19 patients.

Scientists have, over the years, explored sustainable ways of extracting blood from the crabs, worked with fishermen and local communities to rescue and release strays back into the sea and to conserve their habitats

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The reagent market is not an easy one to disrupt and Subita Srimal will vouch for this. “India can make the reagents from biological and synthetic sources for a fraction of the cost of a LAL kit (around Rs 400). It is simple enough,” says Srimal, who set out to make reagents from Indian horseshoe crabs in the 1990s. It was with the support of the Technology Development Board under the Department of Science and Technology and the encouragement of the late neurochemist Bimal Kumar Bacchawat that she incubated her company, Manukirti Biogens, at the Indian Institute of Science’s Molecular Biochemistry Unit, and later, ran it out of a small industrial unit in Jayanagar, south Bengaluru, with a field station in Gangasagar, West Bengal, to harvest crab blood. The product, called the ‘CAL’ test, named after the mangrove crab, hit the market in 1998. “I spent two years trying to get the product included in the Indian pharmacopoeia. It wasn’t easy. We launched 10 kits at Rs 1,500 and sent samples to all major pharma companies. Several government institutes switched to our product but we didn’t have much success with pharma players. And being a single-product company dependent on fishermen, not trawlers, for commercial production, we lost steam. To invest in a recombinant protein method would have pushed up our costs by a lot, so we eventually wound up,” says Srimal, who is now a consultant and partner at ProGrow Pharma Partners.

For a company producing a recombinant protein alternative, the number of gram-negative organisms to be included in the testing alone can be daunting. But horseshoe crab blood, as Chatterji says, “is tried and tested, period”. “It shouldn’t matter if it is the Indian horseshoe crab or the American one. And the one thing the organism has been good at is survival.”

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