The Bacterial

Endotoxins Test

Limulus Amebocyte Lysate Test

A Reference Guide

By Bio Solutions. B-14, Kanika Co-Op. Society, Sarvapally Radhakrishnan Marg, Off Nagardas Road, Near Bhuta High School, Andheri (East),Mumbai – 400069. TELEFAX: 0091-22-26824494. Phone: 9819973583. mail@biosolutions.co.in, biosolutions@rediffmail.com

Introduction

History, Horseshoe

crab &

Endotoxins

INTRODUCTION

The most significant application of the Limulus Amebocyte Lysate (LAL) test is evaluation of parenteral drugs and medical devices for endotoxins [Lipopolysaccharides (LPS)] content. The use of LAL to detect and control the presence of pyrogenic substances in pharmaceuticals and medical devices is a relatively recent development. Pyrogens, endotoxins and lipopolysaccharides are words that are interchangeably used when referring to pyrogen testing. The word pyrogen applies to any material which induces fever. It describes an in vivo characteristic. A solution that contains a fever producing substance is said to be pyrogenic. The word endotoxin refers to a specific pyrogen associated with the membrane of gram negative bacteria. Endotoxins are the most prevalent pyrogen found in aqueous solutions. Lipopolysaccharides LPS describes the biochemical structure of endotoxin. Endotoxins contain a fatty acid portion termed Lipid A and a long chain of repeating sugars or polysaccharides. LPS are purified endotoxins and are used as standards for in vivo pyrogen tests or in vitro assays. Biochemistry The biochemical basis for the use of Limulus amebocyte lysate in the detection of endotoxin lies entirely in the coagulation reaction inherent in Limulus blood. The ability of Limulus blood to from a gelatinous clot was initially described by Howell, Lobe, and Blanchard. These early investigations showed that coagulation was induced by foreign substance and that the circulating amebocyte (= granulocytes) was involved in the reaction. The association between endotoxin and coagulation began in 1956, when Bang reported that infecting the horseshoe crab, Limulus polyphemus, with Vibrio, a Gram-negative bacteria, caused a fatal intravascular coagulation. By 1964, Levin and Bang had demonstrated the extracts of the circulating amebocytes would gel in the presence of Gram-negative bacterial endotoxin. Limulus blood has always held a certain fascination because of its bluish appearance. The blue color is due to hemocyanin, a copper-based oxygen acceptor. The hemocyanin while produced by the cyanocytes, is extra-corporeal and remains in the plasma after centrifugation of whole blood. The amebocyte is the only cell present in Limulus blood. It represents a homogeneous population of nucleated, disk-shaped granular cells. The amebocyte contains all the components of the entire coagulation systems. Cell-free hemolymph does not gel in the presence of endotoxin and does not enhance the endotoxin-mediated reaction associated with amebocyte extracts. Lysates from washed amebocytes have been prepared by lysis in hypotonic solutions by physical disruption using mechanical shear, or by freeze-thaw cycles. The sensitivity of amebocyte lysate to endotoxin can be increased by subsequent extraction with organic solvents. A simplified description of the Limulus coagulation reaction sequence was presented by Young et al. They fractionated amebocyte lysate into tow active fractions using gel filtration chromatography. Fraction I, the column void, contained a heat-labile component and represented material > 75 kDa. Fraction II represented a heat-stable component of lower molecular weight. A mixture of both fractions was necessary for the regeneration of the reactivity to endotoxin seen in unfractionated lysate. Incubation of endotoxin with fraction I

material prior to the addition of material from Fraction II greatly accelerated the time course of coagulation. This supported the two-step mechanism originally proposed by Levin and Bang. Namely, proenzyme (Fraction I) in the lysis is activated by the presence of endotoxin. This active enzyme subsequently catalyzed the conversion of a clotting protein (Fraction II) into an insoluble gel. The rate-limiting reaction is that of endotoxin and the proclotting enzyme with the concentration of active clotting enzyme formed being proportional to the initial concentration of endotoxin. The coagulation reaction is not as simple as originally proposed. It exists as a multicomponent cascade, which is initiated by endotoxin and terminates with gelation. The majority of the biochemical analysis has been performed by Japanese workers using lysate from another species of horseshoe crab. Tachypleus tridentatus, but their observations are thought to apply to Limulus as well. The first coagulation factor, Factor C directly interacts with endotoxin to from Factor C. An anti LPS factor, found in both Limulus and Tachypleus, inhibits the interaction of Factor C and endotoxin. Factor C possesses enzymatic activity, which activates Factor B. Factor B, the active form of Factor B, is then responsible for activating the proclotting enzyme. Factors B and C have not been directly demonstrated in Limulus polyphemus: however Nakamura and Levin have described and activator of the proclotting enzyme and protease N in Limulus. The exact relationship of these Limulus proteins to Factor B and C of Tachypleus is unknown it present. The clotting protein, coagulogen, is the last component of the coagulation cascade. It comprises almost half of total protein present in amebocyte lysate. The active clotting enzyme is a serine protease and hydrolyzes a single peptide bond within the coagulogen to from a shorter peptide, coagulin. Once generated, the coagulin self-associates, forming a three-dimensional lattice structure and eventually gels. The catalytic nature of each activated component in the coagulation cascade serves to amplify the next step in turn. This amplification most probably results in the extreme sensitivity of Limulus amebocyte lysate to endotoxin. All commercial lysates are capable of detecting picogram (pg) quantities (10 -12) of endotoxin. In addition to the endotoxin-mediated cascade which activates the proclotting enzyme, another protein component. Factor G appears to represent an alternative coagulation pathway. Factor G is present in both Tachypleus and Limulus and is activated by B-1,3-D-glucans. Factor G directly activates the proclotting enzyme, which in turn causes the gelation of coagulogen.

ACTIVATION OF ENZYMATIC PATHWAY

BY ENDOTOXINS

Endotoxins

Factor C Factor C (active)

Factor B Factor B (active)

Proclotting Enzyme Clotting Enzyme

Coagulogen Coagulin

Enter Limulus and an MBL scientist named Fred Bang.

Bang was studying the circulation of blood using horseshoe crabs when he found that one of his crabs died as a result of a Vibrio bacterial infection. The infection caused a strange disease in which almost the entire blood volume of the crab clotted into a semi-solid mass. Other bacteria had not produced this sort of reaction at all. Bang began to investigate further and found that only gram-negative bacteria produced this reaction. Furthermore, heat-treated bacteria (dead bacteria) continued to produce the reaction so it wasn't a pathological disease but something different.

Bang noted that the reaction he was observing was very similar to a well-known endotoxin reaction in mammals, the Schwartzman reaction. Back at Johns Hopkins University, he pushed to have this new phenomenon researched more intensively. Jack Levin, a hematologist, joined Dr. Bangs laboratory. What they eventually found was the "fire alarm" system that could be used to detect, with exquisite sensitivity, the fever-producing endotoxins that are so dangerous to people.

Limulus lives in an aquatic world; the sea. The sea is almost literally awash in gram-negative bacteria. Millions can be found in a single gram of sediment. Bacteria that are both harmless as well as pathogenic (disease-causing).

This section explains how an MBL scientist discovered some amazing properties in horseshoe crab blood that enable has revolutionized ways to detect potentially lethal bacterial toxins and spawned a multi-million dollar industry.

Limulus is an arthropod, a close relative to spiders. In fact more closely related to spiders than to true crabs. Arthropods possess a semi-closed circulatory system. We mammals have

literally thousands of miles of blood vessels that carry blood to our tissues through vast networks of capillaries. Bacteria entering our bodies through these capillaries are initially limited in the area they can infect, having to fight their way into the body through these narrow channels, all the while in contact with the white blood cells that are our first line of defense.

The circulatory system of Limulus is far more open. Large sinuses exist that allow blood direct contact with tissues. There are many wide open spaces and bacteria entering a crack in the shell of a horseshoe crab have easy access to large internal areas of the crab, a potentially deadly scenario. Over the course of it's hundreds of millions of years of interacting with the bacterial swarms it coexists with, Limulus, like us, has developed exquisitely sensitive means for detecting the presence of bacteria through the LPS they shed into their environment.

Limulus is cold-blooded. It can't raise it's body temperature to kill off an infection. Nor does it have the vast confusing network of blood vessels to contain an infection. It needs to act quickly, and sometimes even rashly. The soldiers of the immune system in Limulus are it's single type of blood cell, the amoebocyte. As it's name implies it is an amoeboid cell (it has motility). The cell itself is often obloid in the blood stream and perform most of the normal functions associated with blood cells, engulfing foreign or dead cells, transport and storage of digested materials, repair of wound sites, etc. The cells appear oval when seen inside a living crab and they are packed with small granules. These granules contain clotting factors that are released outside the cell when it detects the bacterial endotoxin. When the hemocyte is in the presence of endotoxin it changes dramatically, so much so that it was originally believed Limulus had several types of blood cells. The compact shape changes to an irregular amoeboid shape with numerous cytoplasmic processes streaming in all directions. The cell discharges the granules of coagulogen which empty the cell.

It's a very sensible system. Imagine a horseshoe crab has sustained a small injury. Seawater comes into contact with the tissue and bacteria come into contact with the blood and begin to enter (ie infect) the body of the crab. Small bits of the cell wall slough off as the bacteria propel itself through the blood. A Limulus blood cells detects this tiny fragment and responds by releasing the contents of the granules into the surround medium. These granules contain a clotting factor, called coagulogen. The thought is that by clotting the

Why is horseshoe crab blood blue?

The oxygen-carrying pigment in horseshoe brab blood is a protein called hemocyanin. It is very similar to the hemoglobinmolecule we have in our blood. Hemoglobin gets it's red color (which makes our blood red) from the iron molecule in the center of the protein. Hemocyanin contains a copper molecule which results in a blue color.

immediate surroundings very quickly, the invading bacteria can become enmeshed and therefore stopped! Larger clots may not only stop enmeshed bacteria but serve as a barrier to the outside environment in the case of a severed limb or large incision. Bang found these clots to be very stable and prevented even Brownian motion in trapped bacteria.

The bottom line to all this is that Limulus contains an exceeding sensitive means to detect the presence of bacterial endotoxins that can be detected by the formation of a gel-like clot. This may not strike one as significant until one understands the impact of endotoxins on our health and healthcare systems.

Anything that goes into your body during surgery, by injection, or for therapy, has to be free of bacteria. If not, the recipient will get an infection. Not only must this material be sterile (meaning no living bacteria are present) but it must be pyrogen-free.! As was demonstrated long ago, our bodies, like the bodies of horseshoe crabs , respond to the presence or endotoxin, not just the bacteria. The industry of ensuring that injectable drugs, irrigation fluids, surgical tubing, etc are free of bacterial endotoxins is a big business. In the past, companies maintained large rabbit colonies. Rabbits, like use, are sensitive to endotoxin and if a suspect sample of saline injected into a rabbit caused a fever then it was contaminated. No fever, no contamination. This method was not only expensive (it isn't cheap to keep thousands of rabbits) it is also slow. A rabbit test might require 48 hours to obtain a result. A Limulus amoebocyte lysate (LAL) assay can take as little as 45 minutes. A suspect sample is mixed with reconstituted LAL and allowed to sit in a small tube. After 45 minutes the tube is inverted and if a clot has formed it will stick to the top of the inverted tube.

LAL is a multi-million dollar business. It received FDA approval in the 1970's for use in the testing of drugs, blood products, intravenous fluids, and disposable pharmaceutical devices and in 1983 was registered in the U.S. Pharmacopeia. The lysate is produced by extracting blood from the crab. This is done using a non-lethal method where blood is taken from a large dorsal blood sinus, the pericardium. The crabs are returned to the water within 24 hours and completely recover.

The blood is a milky-blue color due to the copper-pigmented hemocyanin molecule which turns blue upon contact with oxygen the same way our blood turns red. The blood is a mixture of liquid serum and suspended amoebocytes. Of course, the conditions for extracting blood must be sterile and pyrogen-free else the amoebocytes would immediately do their job and form a clot. If these conditions are met, the blood can be centrifuged and the result is a separation of blood cells from the serum. A small whitish pellet forms at the bottom of the tube. Technicians pour off the serum and rinse the pellet with saline. It is then resuspended and added to the collection of amoebocytes. Eventually pyrogen-free, distilled water is mixed with the suspension of blood cells. This causes the cells to absorb fresh water and balloon until they eventually burst (or "lyse" - hence "lysate"). This releases the coagulogen into solution.

The resultant solution is filtered to remove cellular debris and then freeze-dried to form a white powder of the lysate. This lysate is then packaged and sold to be reconstituted as the assay described above.

A few decades ago, there was a bounty on Limulus as it was perceived to be a threat to the shellfish industry. The work of Bang and the resultant market developed by Watson and others has turned this animal into a valued resource. Not only is this commodity renewable and sustainable but the methods are non-lethal to the animal as well. This is a good example or basic research providing additional leverage in the conservation of Limulus and the aquatic environments it inhabits. Who knows where the next discoveries may lead. The line between basic research and applied science is indistinct and quite often it is the unexpected discoveries that are the most rewarding.

Jack Levin demonstrates the removal of blood from a Limulus. This procedure does not permanently harm the animal.

References used in this section

1. -Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor," Biology of Horseshoe Crabs, p.334

2. -Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor," Biology of Horseshoe Crabs , p.334

3. -Segukuchi, Koichi, 1988, "Hemocytes and Coagulogen, A coagulation factor," Biology of Horseshoe Crabs , p.338

4. Mürer, E.H., Levin. J. and Holm, R., 1975. Isolation and studies of the granules of the ameobocytes of Limulus polyphemus, the horseshoe crab. J. Cell Physiol., 86: 533-542

5. Armstrong, P.B. 1979, Motility of the Limulus Amebocyte, Biomedical Applications of the Horseshoe Cran (Limulidae), 73-92.

Quigley, J.P., Corcoran, G., Armstrong, P.B., A Hemolytic Activity Secreted by the Endotoxin-Challenged Horseshoe Crab: A Novel Immune System Operating at the Surface of the Carapace. , Biological Bulletin, 193: 273 (October 1997)

6. Milne, Edwards, H., Historie naturelle des Crustacea., Paris, 1834-40

7. Milne, Edwards, H., L'Anatomie des Limules, 1873

8. Sargent, William., The Year of the Crab., W.W. Norton & Company 1987

THE HORSESHOE CRAB, Limulus polyphemus A “living fossil”

Not a true crab, the horseshoe crab is actually more closely related to spiders, scorpions, and ticks than to other crustaceans. This "living fossil" has been around since well before the dinosaurs. Its large eyes and its unique blood have made it uniquely valuable for biomedical research.

Spring high tides trigger a massive migration of the Atlantic genus, Limulus polyphemus onto sheltered Delaware and New Jersey beaches. The sand can be piled deep with spawning crabs, and females can lay as many as 88,000 eggs each season. Migratory shorebirds rely on these abundant eggs to fuel their journey from South America to the Arctic Circle.

However, horseshoe crabs have become valuable as bait for commercial fishermen, who can catch tens of thousands in one day. Since 1990, the horseshoe crab population of the Delaware Bay has been reduced by half, and the number of shore birds has also greatly declined. Scientists, biologists, and fishermen are now working to restore a balance that has been in place for millions of years. A Very Ancient History Not a true crab, the horseshoe crab is actually more closely related to spiders, scorpions, and ticks than to other crustaceans. This large marine arthropod gets its common name from its shell, or carapace, which is U-shaped. Brownish-gray in color, the carapace provides camouflage against the muddy or sandy seabed where the horseshoe crab lives.

They haven't changed much since the Devonian era, some 360 million years ago, well before dinosaurs. Fossil data suggests that there were never more than twenty species of horseshoe crabs. Today only four species, grouped into three genera, remain.

Two genera are found along the coast of Southeast Asia and nearby countries such as Japan. The third is found along the entire Atlantic coast of the United States and along the Gulf of Mexico as far as the Yucatan. Believed to have once been far more widely distributed, Limulus is the only surviving species in its genus, which indicates that its lineage is very ancient. The scientific name for the Atlantic coast crab is Limulus polyphemus, after the one-eyed giant of Greek myth.

Because its basic body design has changed so little over the ages, the horseshoe crab is often referred to as a "living fossil.” They have as much genetic variation as many other species, but are so well adapted to their ecological niche that large-scale species radiation simply hasn't occurred. As biologist Mark Botton of

Fordham University puts it, "They seem to have hit upon a body plan that worked a long time ago, and hasn't really changed."

A Curious Body

The body of this extraordinarily well-adapted animal has highly unusual features. Though named after a one-eyed character, Limulus (meaning "sidelong") actually has ten eyes: one oval lateral eye on each side of its shell, two small ones in the center, five light-receptive organs beneath its shell, and one in its tail. The larger eyes are compound, like those of insects, allowing the animal to see in all directions.

Unlike true crabs, which have five pairs of legs, horseshoe crabs have four. First come a pair of appendages called "chelicerae," then a pair of "pedipalps," followed by the four pairs of legs. These eight legs have pincers, and are used for walking and for grinding and manipulating food. To find its favorite foods--worms, mollusks, and dead fish--the horseshoe crab crawls along the bay bottom using its pedipalps as feelers to detect prey. When it comes upon a worm or clam, the small claws pick it up and move it to the bristly areas near the base of the walking legs where the mouth is located. The horseshoe crab has no jaws and uses the bristles to crush the food as it moves its legs.

Behind the horseshoe crab’s mouth and the walking legs is its abdomen, which is connected to the rest of the carapace by a flexible joint that allows it to move up and down. The gills, which are called book gills and are also found in spiders, are attached to the underside of the abdomen. Made up of about one hundred thin leaves or plates, these respiratory organs enable the animal to get oxygen from the water, and also from the air (if the crab is on the beach) as long as the gills are wet. Last is a long tail, or telson, which helps the animal flip over if turned upside down. The tail has no stinger and is not poisonous. The tail will not regenerate if broken off, so a horseshoe crab should always be picked up by the shell. Though they may look dangerous, horseshoe crabs are completely harmless.

Young horseshoe crabs can swim upside down. To propel themselves, they flap their gills and move their abdomens up and down.

Breeding Crabs Once Covered Beaches Meters Deep During the cold months, Limulus lies buried in the bottom of the Delaware Bay and the Atlantic Ocean. In the spring, a signal synchronized in some way with spring high tides triggers a massive migration. By late May, millions of these ancient creatures begin crawling shoreward in the Delaware Bay, seeking out sandy beaches that are protected from the waves.

At peak times, beaches can be piled deep with crabs and the air filled with the sound of shells clacking against each other. Peak spawning usually coincides with the high tides that accompany the full and new moons in May and June, and generally takes place at night. Water temperature and weather conditions such as heavy surf can prevent spawning.

Males come in to shore attached to females, or alone, and accumulate at the base of the beach. Studies have shown that males use their eyes to find mates.

The males, which are smaller than the females, have special pincers at the end of their first pair of walking legs with which they attempt to hook onto the female's abdomen. Sometimes a chain of several crabs is formed, as one male clasps onto another behind a female in a tenacious embrace called "amplexus." Once a male is in tow, the female slowly makes her way to the edge of the water, where she scoops out a nest six to eight inches (about fifteen to twenty centimeters) deep in the sand. There she deposits thousands of BB-sized eggs, while the male passes over and fertilizes the eggs. As many as a dozen males may jostle around the mating pair.

This form of external fertilization makes horseshoe crabs unique among arthropods. Several nests may be dug during a single beach trip, and females may make additional trips on subsequent tides. Studies in Delaware found that females laid an average of 3,650 eggs per nest, and can lay as many as 88,000 eggs per season.

If nests are numerous, female crabs may disturb earlier nests when digging new ones. The disturbed eggs accumulate on the surface of the beach, providing a feast for shore birds, which cannot reach the buried eggs.

By spawning at full and new moons, when tides are highest, the female protects her eggs from being washed away. Nests are usually located close enough to the water to stay damp, but high enough for the sand to contain adequate oxygen. These factors, along with the temperature, determine how long it takes for the eggs to develop. Eggs usually hatch in about a month, and out come tiny horseshoe crabs--minus tails--about three millimeters long. They're called trilobite larvae because they look so much like ancient, extinct trilobites.

Molting When a horseshoe crab outgrows its shell, it has to molt: leave the old shell and grow a new one. The old shell splits around the front edge and the crab crawls out. The new shell, which is about 25 percent larger, soon hardens. Shells found on the beach are typically these outgrown ones.

Horseshoe crabs molt five times in their first year of life, two or three times during their second year, twice in the third year, and then once a year until they mature at nine or ten years of age.

For their first two years, juveniles tend to stay in the intertidal flats near where they hatched, then gradually move to deeper waters. Feeble swimmers, adult horseshoe crabs walk on the ocean floor. For most of the year they crawl along the bottom of bays and along the continental shelf. Once they reach maturity, they will crawl back to shore each year to spawn.

They have few natural predators, though loggerhead turtles sometimes tear out the flapping gills and pieces of the horseshoe crab have been found inside sharks. No one really knows how long horseshoe crabs can live. A few have been kept in aquaria for as long as fifteen years.

Useful Horseshoe Crab Useful in many ways, the horseshoe crab has proven uniquely valuable to basic biomedical science.

Crab Blood Could Save Your Life

The blood (hemolymph) of Limulus turns blue when exposed to oxygen, and turns out to be perhaps the most valuable part of this ancient creature, from a human point of view. When a crab receives a wound, cells form a clot, and kill certain kinds of bacteria which are also harmful to humans. This process was discovered in the early 1950s by a scientist named Frederick Bang, who was able to separate the chemical that caused the bacteria-sensitive clots to form. Later, the extract was named LAL (Limulus Amoebocyte Lysate), and it is used to detect whether things that go into the human body--including injectable drugs, needles, and heart valves--are free of dangerous endotoxin producing bacteria. Using LAL is more accurate, simpler, and less expensive than similar tests for bacteria.

Blood is collected from horseshoe crabs taken out of the shallow waters off the Atlantic coast, during the summer months.

They're checked for health and then bled through a stainless steel tube. It takes around five minutes to extract about 20 percent of the animal's blood, after which it is returned to the ocean. About 90 percent of horseshoe crabs survive this procedure. Approximately two hundred thousand crabs are bled each year for LAL.

Educational Eyes

For over fifty years, the eyes of horseshoe crabs have been used in eye research. The crab's lateral compound eye has shown scientists a great deal about how the human eye functions. It's easy to study because both the horseshoe crab's eye and its optic nerve (which transmits signals from the eye to the brain) are large, and because its organization is much simpler than a human's. This has enabled scientists to analyze the electric signals that send visual information to the brain, and therefore to understand many underlying principles of all visual systems--how the human eye perceives lines, borders, and contrasts, for example. Research on horseshoe crabs has also helped researchers understand diseases such as retinitis pigmentosa, which causes tunnel vision and can lead to blindness. In fact, the horseshoe crab is the only animal for which we now understand the complete neural code for vision, which should help us understand more complex visual systems in the future.

An Age-Old Survivor The horseshoe crab, Limulus polyphemus, has been around for a very long time. Fossils from British Columbia (situated at the time in warm, shallow tropical seas) show that relatives lived in North America some 520 million years ago. The horseshoe crab went on to survive several mass extinctions, including ones that wiped out many other marine species. The habitat of this genus of the ancient animal is now restricted to a little peninsula on the Eastern shore of the United States, where it has been spawning since time immemorial in sequence with the phases of the moon. It now faces arguably the most serious crisis ever, one that is man-made.

. . . Until Now?

Between the 1880s and the 1920s about a million horseshoe crabs were harvested each year for use as fertilizer and in hog fodder. Chemical supplements replaced them, but fifty years passed before the crab population rebounded.

Horseshoe crabs have again become a valuable commodity, this time as bait. Traditionally fishermen picked crabs off the beaches by hand, for free, and the harvest was small. However, the domestic and international market for eels and whelks (also called conch) has been growing rapidly. Female, egg-bearing horseshoe crabs make particularly good bait, so the demand for Limulus has greatly increased as well. With blue crabs scarce and other fisheries in a slump, horseshoe crabs have been selling for between eighty-five cents and a dollar a piece, a price that has made it even more attractive as bait to commercial fishermen and others. Some fishermen converted their boats to trawlers, which drag nets across the ocean floor and can catch tens of thousands of horseshoe crabs in one day.

A solution might lie in a synthetic horseshoe crab "scent" that would replace the need to use actual animals as bait, which scientists are working on.

Since 1985, the commercial harvest of horseshoe crabs along the Atlantic coast has greatly expanded. From 1990 to 1994, the National Marine Fisheries Service reported the Delaware, Maryland, and New Jersey harvest increasing from 685,648 pounds (311,000 kilograms) to 1,386,367 (628,846 kilograms), which translates to an annual haul estimated at half a million crabs. Dr. Mark. L. Botton, associate professor of biology at Fordham University, cites a much higher figure: "It's now becoming clear that the number taken is approaching a million a year." Botton, who is studying the New Jersey crab population, notes that "on the one hand, some of the watermen argue that there's absolutely no indication that the numbers are going down. On the other hand, some of the environmental spokespeople are talking about the population on the verge of extinction. Neither of these extremes is correct, and we're trying to provide the data that allow for proper management of the population."

This species matures slowly, and doesn't begin to reproduce until around age ten. It's also long-lived. These factors lessen the effect of a single or several poor spawning years, so horseshoe crab populations tend to be fairly stable. It

also means that serious, long-term impacts on the population will take a decade to become fully apparent.

Habitat Degradation

It is the absence of inlets or other breaks in the Delaware Bay shorefront that makes it an ideal habitat for horseshoe crabs. Environmentalists and researchers now believe that much of the recent population decline can be attributed to severe habitat degradation on the New Jersey side of the Delaware Bay, in the form of man-made inlets and sandbars. These artificial breaks reduce beach sand deposits, and large areas of mud flats form behind the breached shorefronts. Huge numbers of horseshoe crabs--three hundred thousand by some estimates--have been observed entrapped in these back-marsh mud areas. The adult crabs are stranded and die, and their eggs do not develop in the mud.

Ninety percent of the horseshoe crab stock is located between Virginia and New Jersey, and only five years ago prime beaches would have been literally piled meters deep with spawning horseshoe crabs. Spawning surveys are not always reliable, but the effect of habitat degradation is becoming more and more obvious. Four separate scientific studies conducted in Delaware Bay since 1990 have estimated that the horseshoe crab population has declined by more than 50 percent. By any count it is apparent that the Delaware Bay population of Limulus polyphemus is swiftly declining.

Fewer Horseshoe Crabs Means Less For Other Species

Like all species, the horseshoe crab is part of a web of life, the disruption of which affects many other kinds of animals:

• Migratory shorebirds rely on abundant crab eggs to fuel their northward journey. These include red knots, sanderlings, ruddy turnstones, and sandpipers. Good stopover sites for these migrating birds are few, and no substitute exists for Delaware Bay.

It could take years for the crab numbers to rebound, during which time the bird populations will be highly vulnerable.

• The highly nutritious eggs are also eaten by raccoons, foxes, diamondback terrapins, moles, and even mollusks.

• Fish feed on juvenile horseshoe crabs and on recent molts.

A steep decline in the horseshoe crab population will have a significant impact on the fishing and medical industries, as well as on ecotourism. Birdwatchers who flock to the Delaware Bay each spring to see the feeding shorebirds bring in millions of dollars to local businesses.

What's Being Done

In 1995, a large and unusual coalition of environmental groups and businesses called for emergency regulations on horseshoe crab fishing. In response, the New Jersey Department of Environmental Protection restricted hand-harvesting to two nights a week.

Trawling--dragging nets across the ocean floor--continued, however, until the Governor of New Jersey, Christine Whitman, issued a temporary ban on all trawling and hand-harvesting of horseshoe crabs in May 1997. The ban was subsequently struck down, then reversed in an out-of-court settlement with the New Jersey Audubon Society and the American Littoral Society and reinstated.

In Maryland, trawling and dredging are prohibited between April 1 and June 30 within Chesapeake Bay, coastal bays, and within one mile (1.6 kilometers) of the Atlantic Ocean. Hand collection is also limited during this period. Virginia has banned trawling with state waters and within three miles (4.8 kilometers) of the coast, and requires commercial fishermen to report any crabs harvested as bycatch.

New Jersey has dedicated $80,000 to research the population size of the local horseshoe crab population, and also that of migrating shorebirds.

Maryland and Virginia are also taking steps to learn more about the species and to protect it, especially during spawning. Maryland began a spawning survey in 1994, and also tags horseshoe crabs. The hope is that a balance--a sensible harvest--can be arrived at. "While I'm concerned that the trends are downward, I don't think it's cause for panic yet," says Dr. Botton. "What I do think is called for is some prudent management strategy to conserve enough crabs to keep the shorebirds fed, but yet permit the watermen to have a livelihood."

Efforts in Japan

Another genus of horseshoe crabs is native to Japan. Along with over a dozen other aquatic species, it has been declared endangered. Land reclamation and water pollution are the culprits. The animal used to thrive in large areas around the Seto Inland Sea and northern Hyushu, in particular on a tidal flat along Kasaoka Bay. Although it was designated a protected area, in 1966 the Japanese government began a huge land reclamation project here. Completed in 1990, the project has completely wiped out the local crab population.

In 1975. the Japanese government established the Horseshoe Crab Protection Center. When the drastic effect of the reclamation project became apparent--the estimate is that only two thousand to four thousand Japanese horseshoe crabs are left--the center was turned into the Kasaoka City Horseshoe Crab Museum. In 1993, the museum launched a five-year project to raise horseshoe crabs from eggs for release into the bay.

BACTERIAL ENDOTOXINS Endotoxins are part of the outer cell wall of bacteria. Endotoxins are invariably associated with Gram-negative bacteria as constituents of the outer membrane of the cell wall. Although the term endotoxin is occasionally used to refer to any "cell-associated" bacterial toxin, it should be reserved for the lipopolysaccharide complex associated with the outer envelope of Gram-negative bacteria such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, and other leading pathogens.

The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). Toxicity is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide components. The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS. LPS elicits a variety of inflammatory responses in an animal. Because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of Gram-negative bacterial infections.

The relationship of endotoxins to the bacterial cell surface is illustrated in Figure 1 below.

Figure 1. Structure of the cell surface of a Gram-negative bacterium

Gram-negative bacteria probably release minute amounts of endotoxin while growing. For example, it is known, that small amounts of endotoxin may be released in a soluble form, especially by young cultures. However, for the most part, endotoxins remain associated with the cell wall until disintegration of the bacteria. In vivo , this results from autolysis of the bacteria, external lysis mediated by complement and lysozyme, and phagocytic digestion of bacterial cells.

Compared to the classic exotoxins of bacteria, endotoxins are less potent and less specific in their action, since they do not act enzymatically. Endotoxins are heat stable (boiling for 30 minutes does not destabilize endotoxin), but certain powerful oxidizing agents such as superoxide, peroxide and hypochlorite, degrade them. Endotoxins, although strongly antigenic, cannot be converted to toxoids. A comparison of the properties of bacterial endotoxins and classic exotoxins is shown in Table 1.

Table 1. Characteristics of bacterial endotoxins and classic exotoxins.

PROPERTY ENDOTOXIN EXOTOXIN

CHEMICAL NATURE Lipopolysaccharide(mw =

10kDa) Protein (mw = 50-

1000kDa)

RELATIONSHIP TO CELL

Part of outer membrane Extracellular, diffusible

DENATURED BY BOILING

No Usually

ANTIGENIC Yes Yes

FORM TOXOID No Yes

POTENCY Relatively low (>100ug) Relatively high (1 ug)

SPECIFICITY Low degree High degree

ENZYMATIC ACTIVITY No Usually

PYROGENICITY Yes Occasionally

Lipopolysaccharides participate in a number of outer membrane functions that are essential for bacterial growth and survival, especially within the context of a host-parasite interaction. An intact outer membrane exerts several vital functions in Gram-negative bacteria:

1. It is a permeability barrier that is permeable only to low molecular weight, hydrophilic molecules. In the Enterobacteriaceae, the ompF and ompC porins exclude passage of all hydrophobic molecules and any hydrophilic molecules greater than a molecular weight of about 700 daltons. This prevents penetration of the bacteria by bile salts and other toxic molecules from the GI tract. It also retains periplasmic components.

2. It impedes destruction of the bacterial cells by serum components and phagocytic cells.

3. It plays an important role as a surface structure in the interaction of the pathogen with its host. For example, LPS may be involved in adherence (colonization), or resistance to phagocytosis, or antigenic shifts that determine the course and outcome of an infection.

Chemical Nature of Endotoxin.

Most of the work on the chemical structure of endotoxin has been done with species of Salmonella and E. coli. LPS can be extracted from whole cells by treatment with 45% phenol at 90o. Mild hydrolysis of LPS yields Lipid A plus polysaccharide.

Lipopolysaccharides are complex amphiphilic molecules with a mw of about 10kDa, that vary widely in chemical composition both between and among bacterial species The general architecture of LPS is shown in Figure 2. The general structure of Salmonella LPS is shown in Figure 3 and the complete structure of Salmonella lipid A is illustrated in Figure 4.

Figure 2. General architecture of Lipopolysaccharide

Figure 3. General Structure of Salmonella LPS

Glc = glucose; GlcNac = N-acetyl- glucosamine; Gal = galactose; Hep = heptose; P = phosphate; Etn = ethanolamine; R1 and R2 = phoshoethanolamine or aminoarabinose. Ra to Re indicate incomplete forms of LPS. The Rd2 phenotype (not shown) would have only a single heptose unit. The Rc, Rd2, and Rd1 mutants lack the phosphate group attached to Hep.

Figure 4. Complete structure of the Lipid A Moiety of LPS of S. typhimurium, S. minnesota, and E. coli

LPS consists of three components or regions:

• Region I. Lipid A • Region II. Core (R) antigen • Region III. Somatic (O) antigen or O polysaccharide

1. Lipid A is the lipid component of LPS. It contains the hydrophobic, membrane-anchoring region of LPS. Lipid A consists of a phosphorylated N-acetylglucosamine (NAG) dimer with 6 or 7 fatty acids (FA) attached. Usually 6 FA are found. All FA in Lipid A are saturated. Some FA are attached directly to the NAG dimer and others are esterified to the 3-hydroxy fatty acids that are characteristically present. The structure of Lipid A is highly conserved among Gram-negative bacteria. Among Enterobacteriaceae Lipid A is virtually constant.

2. Core (R) polysaccharide is attached to the 6 position of one NAG. The R antigen consists of a short chain of sugars. For example:KDO - Hep - Hep - Glu - Gal - Glu - GluNAc -

Two unusual sugars are usually present, heptose and 2-keto-3-deoxyoctonoic acid (KDO), in the core polysaccharide. KDO is unique and invariably present in LPS and so has been an indicator in assays for LPS (endotoxin).

With minor variations, the core polysaccharide is common to all members of a bacterial genus (e.g. Salmonella), but it is structurally distinct in other genera of Gram-negative bacteria. Salmonella, Shigella and Escherichia have similar but not identical cores.

3. The O antigen or O side chain is attached to the core polysaccharide. It consists of repeating oligosaccharide subunits made up of 3 - 5 sugars. The individual chains vary in length ranging up to 40 repeat units. The O polysaccharide is much longer than the core polysaccharide and it maintains the hydrophilic domain of the LPS molecule. A major antigenic

determinant (antibody-combining site) of the Gram-negative cell wall resides in the O polysaccharide. Great variation occurs in the composition of the sugars in the O side chain between species and even strains of Gram-negative bacteria. At least 20 different sugars are known to occur and many of these sugars are characteristically unique dideoxyhexoses, which occur in nature only in Gram-negative cell walls. Variations in sugar content of the O polysaccharide contribute to the wide variety of antigenic types of Salmonella and E. coli and presumably other strains of Gram-negative species. Particular sugars in the structure, especially the terminal ones, confer immunological specificity of the O antigen, in addition to "smoothness" (colony morphology) of the strain. Loss of the O specific region by mutation results in the strain becoming a "rough" (colony morphology) or R strain.

The structure of LPS in Salmonella typhimurium and E. coli is seen in Figure 3). The elucidation of the structure of LPS relied heavily on the availability of mutants each blocked at a particular step in LPS synthesis. The biosynthesis of LPS is strictly sequential. The core sugars are added sequentially to Lipid A by successive additions, and the O side chain is added last, one preassembled subunit at a time. The properties of mutants producing incomplete LPS molecules suggests the nature and biological functions performed by various parts of the LPS molecule:

1. Loss of the O antigen results in loss of virulence suggesting that this portion is important during a host-parasite interaction. It is known that such "rough" mutants are more susceptible to phagocytosis and serum bactericidal reactions.

2. Loss of the more proximal parts of the core, as in "deep rough" mutants (i.e. in Rd1, Rd2, and Re mutants) makes the strains sensitive to a range of hydrophobic compounds, including antibiotics, detergents, bile salts and mutagens. This area contains a large number of charged groups and is thought to be important in maintaining the permeability properties of the outer membrane.

3. Mutants in the assembly of Lipid A cannot be isolated except as conditional lethal mutants and this region must therefore be essential for cell viability. The innermost region of LPS, consisting of Lipid A and three residues of KDO, appears to be essential for viability, presumably for assembling the outer membrane.

LPS and virulence of Gram-negative bacteria

Both Lipid A (the toxic component of LPS) and the polysaccharide side chains (the nontoxic but immunogenic portion of LPS) act as determinants of virulence in Gram-negative bacteria. Virulence and the property of "smoothness" (associated with an intact O polysaccharide) are regularly associated in many bacterial infections. The polysaccharide chain must also be important for virulence as shown by the fact that small changes in the sugar sequences in the side chains of LPS, result in major changes in virulence. How are the polysaccharide side chains involved in the expression of virulence? There are a number of possibilities:

a. Smooth antigens could allow organisms to adhere specifically to certain tissues, especially epithelial tissues.

b. Smooth antigens probably allow resistance to phagocytes, since rough mutants are more readily engulfed and destroyed by phagocytes.

c. The hydrophilic O polysaccharides could act as water-solubilizing carriers for toxic Lipid A. It is known that the exact structure of the polysaccharide can greatly influence water binding capacity at the cell surface.

d. The O antigens could provide protection from damaging reactions with antibody and complement. Rough strains of Gram-negative bacteria derived from virulent strains are generally non virulent. Smooth strains have polysaccharide "whiskers" which bear O antigens projecting from the cell surface. The O antigens are the key targets for the action of host antibody and complement, but when the reaction takes place at the tips of the polysaccharide chains, a significant distance external to the general bacterial cell surface, complement fails to have its normal lytic effect. Such bacteria are virulent because of this resistance to immune forces of the host. If the projecting polysaccharide chains are shortened or removed, antibody reacts with antigens on the general bacterial surface, or very close to it, and complement can lyse the bacteria (Thus, "rough" colonial strains are non virulent.).

Biological Properties of Endotoxins

Endotoxins are toxic to most mammals. Even though endotoxins are strong antigens, they seldom elicit immune responses which gives full protection to the animal against secondary challenge with the endotoxin. They cannot be toxoided. Regardless of the bacterial source, all endotoxins produce the same range of biological effects in the animal host.

Most of our knowledge of the biological activities of endotoxins derives not from the study of natural disease but by challenge of experimental animals.

The injection of living or killed Gram-negative cells, or purified LPS, into experimental animals causes a wide spectrum of nonspecific pathophysiological reactions such as:

1. fever 2. changes in white blood cell counts 3. disseminated intravascular coagulation 4. tumor necrosis 5. hypotension 6. shock 7. lethality

Injection of large doses of endotoxin results in death in most mammals. The sequence of events follows a regular pattern: (1) latent period; (2) physiological distress (diarrhea, prostration, shock); (3) death. How soon death occurs varies on the dose of the endotoxin, route of administration, and species of animal. Animals vary in their susceptibility to endotoxin

Since Lipid A is embedded in the outer membrane of bacterial cells, it probably only exerts its toxic effects when released from multiplying cells in a soluble form, or when the bacteria are lysed as a result of autolysis, complement and the membrane attack complex (MAC), ingestion and killing by phagocytes, or killing with certain types of antibiotics. It is thought that LPS released into the bloodstream by lysing Gram-negative bacteria is first bound by certain plasma proteins identified as LPS-binding proteins. The LPS-binding protein complex interacts with CD14 receptors on monocytes and macrophages and other types of receptors on endothelial cells. In monocytes and macrophages three types of events are triggered during their interaction with LPS (See also Handout 11 Figure 5):

1. Production of cytokines, including IL-1, IL-6, IL-8, TNFalpha and platelet-activating factor. These in turn stimulate production of prostaglandins and leukotrienes. These are powerful mediators of inflammation and septic shock that accompanies endotoxin toxemia.

2. Activation of the complement cascade. 3. Activation of the coagulation cascade. During infectious disease

caused by Gram-negative bacteria, endotoxins released from, or part of, multiplying cells have similar effects on animals and significantly contribute to the symptoms and pathology encountered.

The range of inflammatory effects caused by LPS during Gram-negative bacteremia or septicemia are outlined below.

1. Complement activation: C3a and C5a cause histamine release (leading to vasodilation) and effect neutrophil chemotaxis and accumulation. The result is inflammation.

2. Initial activation of Hageman factor (blood-clotting Factor XII), which, in turn, can activate several humoral systems (See Handout 11 Figure 6) resulting in

a. coagulation: a blood clotting cascade that leads to coagulation, thrombosis, acute disseminated intravascular coagulation, which depletes platelets and various clotting factors resulting in internal bleeding.

b. activation of the complement alternative pathway (as above, which leads to inflammation)

c. plasmin activation which leads to fibrinolysis and hemorrhaging.

d. kinin activation releases bradykinins and other vasoactive peptides which causes hypotension. The net effect of LPS is to induce inflammation, intravascular coagulation, hemorrhage and shock.

3. LPS acts as a B cell mitogen stimulating the polyclonal differentiation and multiplication of B-cells and the secretion of immunoglobulins, especially IgG and IgM.

4. LPS activates macrophages to enhanced phagocytosis and cytotoxicity. Macrophages are stimulated to produce and release lysosomal enzymes,

IL-1 ("endogenous pyrogen"), and tumor necrosis factor (TNFalpha), as well as other cytokines and mediators.

These physiological activities of endotoxins are mediated mainly by the Lipid A component of LPS. The primary structure of Lipid A has been elucidated and Lipid A has been chemically synthesized. Its biological activity appears to depend on a peculiar conformation that is determined by the glucosamine disaccharide, the PO4 groups, the acyl chains, and also the KDO-containing inner core. Thus Lipid A is a powerful biological response modifier that can stimulate the mammalian immune system.

The BACTERIAL ENDOTOXINS TEST

Procedure:

Important criteria

• Temperature should be 37 ± 1˚C. • Time 60 minutes ± 2 minutes • Test tube size 10 X 75 mm.

100mL Sample + 100mL LAL

37°C ± 1°C, 60 ± 2 min

Negative Test Viscous/ Cloudy, No gel

10X75 mm tubes

Positive Test Gel formation

Reagents

• Lyophilized LAL Reagent, Sensitivity ( λ EU/mL) • Control Standard Endotoxin (CSE) with CoA • Lysate Reagent Water (LRW), LAL Reagent Water (LRW), BET Water.

Accessories

• Heating Block • Vortex Mixer • Depyrogenated glass test tubes for assay (10X75mm) • Depyrogenated glass test tubes for dilutions • Micro Pipette – 10 - 100µL, 20 - 200µL, 1000µL • Sterile Micropipette Tips • Timer • Depyrogenated glass pipettes - 5mL, 2mL

REGULATORY INITIATIVES LAL Reagent Licensure The Bureau of Biologics (now the Centre for Biologics Evaluation and Research, CBER) elected to regulate LAL reagents as an in vitro biologic because they were a blood product with potential to become a human diagnostic test and a replacement for the Pyrogen Test (U.S. Public Health Service 1977; Weary 1984). LAL reagents were first marketed in 1977 as a licensed biological product, but their use was restricted at that time to in-process testing of parenterals. CBER exerts close scrutiny over the LAL industry through its manufacturing compliance program. LAL Test Guideline After seven years of review, the FDA and the LAL industry agreed on the LAL TEST Guideline (FDA 1987). A pharmaceutical or medical device producer could switch from rabbit to LAL testing if the LAL Test Guideline were followed. The FDA approved LAL because of concern about the relative insensitivity and unreliability of the rabbit test. There was less concern about the remote possibility of missing non-endotoxin pyrogens or materials-mediated pyrogens have not materialized. The FDA’s LAL Test Guideline was the most influential document during the rapid growth of LAL testing in the past two decades. It introduced the concept of the Endotoxin Limits (EL) and provided formulas for dilution (MVD, Maximum Valid Dilution) and concentration limits (MVC, Minimum Valid Concentration). The LAL Test Guideline has detailed sections for testing parenteral drugs and medical devices. Three types of testing are described. First, Initial QC is a set of assays designed to qualify analysts and confirm the label-claim sensitivity of new LAL reagents with calibrated standards. These infrequent tests require assaying one vial of reagent with four replicates from each endotoxin standard dilution, 2λ through 1/4λ, which brackets the expected endpoint of the LAL reagent. For the other two tests, the same standard dilution series (SDS) is required in quadruplicate for validation and in duplicate for routine LAL test. In other words, the test for label claim is accomplished with the SDS Harmonized BET A BET became effective in January 2001 in the 2nd supplement to USP 24 and the European Pharmacopiea, which was produced by the International Conference of Harmonization. The LAL Test Guideline and the new BET are now very similar. The new text includes simplified procedures and inclusion of all LAL methods. In the unlikely case of dispute, the gel-clot method is the referee test.

THE BACTERIAL ENDOTOXINS TEST

INTRODUCTION The Bacterial Endotoxins Test can be performed using lysate extracted form the Amoebocytes of Limulus polyphemus (LAL), Tachypleus tridentatus, Tachypleus gigas (TAL), Carcinoscropius rotundicauda - CAL. USP 29 recognizes LAL and TAL from T. tridentatus spp. The IP 2000 addendum recognizes all LAL, TAL and CAL.

BACTERIAL ENDOTOXINS The test for bacterial endotoxins is used to detect or quantify endotoxins of gram-negative bacterial origin using amoebocyte lysate from horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). There are 3 techniques for this test: the gel-clot technique, which is based on gel formation; the turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate; and the chromogenic technique, based on the development of colour after cleavage of a synthetic peptide-chromogen complex. The following 6 methods are described in the present chapter: Method A. Gel-clot method: limit test Method B. Gel-clot method: semi-quantitative test Method C. Turbidimetric Kinetic method Method D. Chromogenic Kinetic method Method E. Chromogenic end-point method Method F. Turbidimetric end-point method Proceed by any of the 6 methods for the test. In the event of doubt or dispute, the final decision is made based upon method A unless otherwise indicated in the monograph. The test is carried out in a manner that avoids endotoxin contamination. Apparatus Depyrogenate all glassware and other heat-stable apparatus in a hot-air oven using a validated process. A commonly used minimum time and temperature is 30 minutes at 250 ºC. If employing plastic apparatus, such as microtitre plates and pipette tips for automatic pipetters, use apparatus shown to be free of detectable endotoxin and of interfering effects for the test. NOTE: In this chapter, the term ‘tube’ includes all type of receptacles, for example microtitre plate well.

Preparation of the standard endotoxin stock solution The standard endotoxin stock solution is prepared from an endotoxin reference standard that has been calibrated against the International Standard, for example endotoxin standard Biological Reference Preparation (BRP). (EP) or US Reference Standard (USP) Endotoxin is expressed in International Units (IU). The equivalence in IU of the International Standard is stated by the World Health Organisation. NOTE: One International Unit (IU) of endotoxin is equal to one Endotoxin Unit (E.U.) Follow the specifications in the package leaflet and on the label for preparation and storage of the standard endotoxin stock solution. Preparation of the standard endotoxin solutions After vigorously mixing the standard endotoxin stock solution, prepare appropriate serial dilutions of this solution using water for bacterial endotoxin test (water for BET). Use the solutions as soon as possible to avoid loss of activity by adsorption. Preparation of the test solutions Prepare the test solutions by dissolving or diluting active substances or medicinal products using water for BET. Some substances or preparations may be more appropriately dissolved or diluted in other aqueous solutions. If necessary, adjust the pH of the test solution (or dilution thereof) so that the pH of the mixture of the lysate and test solution falls within the pH range specified by the lysate manufacturer. This usually applies to a product with a pH in the range of 6.0 to 8.0. The pH may be adjusted by the use of acid, base or a suitable buffer, as recommended by the lysate manufacturer. Acids and bases may be prepared for concentrates or solids with water for BET in containers free of detectable endotoxin. Buffers must be validated to be free of detectable endotoxin and interfering factors. Determination of the Maximum Valid Dilution The Maximum Valid Dilution (MVD) is the maximum allowable dilution of a sample at which the endotoxin limit can be determined. Determine the MVD using the following formulae: Endotoxin Limit x Concentration of test solution MVD = --------------------------------------------------------------- λ

Endotoxin limit The Endotoxin Limit for active substances administered parenterally, defined on the basis of dose, is equal to: Endotoxin Limit = K / M K = Threshold pyrogenic dose of endotoxin per kilogram of body mass in a single hour period. 5 EU/Kg Body weight for parenteral drugs except those administered intrathecally. 0.2 EU/kg for intrathecal drugs.

The limit formula for radio pharmaceuticals is 175/V except for intrathecally administered products. 14/V for intrathecal drugs. V equals the maximum recommended dose, in mL, at the expiration date or time. For drugs administered on a per Square Meter of Body Surface: 5 EU/ [(dose * 1.8 sq.. m.)/ 70 Kg]

M = Maximum recommended dose of product per kilogram of body mass in a single hour period. The endotoxin limit for active substances administered parenterally is specified in units such as IU/mL, IU/mg. IU/Unit of biological activity, etc., in monographs. Concentration of test solution:

- in mg/mL if the endotoxin limit is specified by mass (IU/mg), - in Units/mL if the endotoxin limit is specified by unit of biological activity (IU/Unit), - in mL/mL if the endotoxin limit is specified by volume (IU/mL)

λ = the labeled lysate sensitivity in the gel-clot technique (IU/mL) or the lowest point used in the standard curve of the turbidimetric or chromogenic techniques.

GEL-CLOT TECHNIQUE (METHODS A AND B)

The gel-clot technique allows detection or quantification of endotoxins and is based on clotting of the lysate in the presence of endotoxin. The concentration of endotoxins required to cause the lysate to clot under standard conditions is the labeled lysate sensitivity. To ensure both the precision and validity of the test, confirm the labeled lysate sensitivity and perform the test for interfering factor as described under 1. Preparatory testing.

1. PREPARATORY TESTING

(i) Confirmation of the labeled lysate sensitivity Confirm in 4 replicates the labeled sensitivity λ, expressed in IU/mL, of the lysate solution prior to use in the test. Confirmation of the lysate sensitivity is carried out when a new batch of lysate is used or when there is any change in the experimental conditions which may affect the outcome of the test. Prepare standard solutions of at least 4 concentrations equivalent to 2λ,λ , 0.5λ and 0.25λ by diluting the standard endotoxin stock solution with water for BET. Mix a volume of the lysate solution with an equal volume of 1 of the standard solutions (such as 0.1 mL aliquots) in each tube. When single test vials or ampoules containing lyophilized lysate are employed, add solutions directly to the vial or ampoule. Incubate the reaction mixture for a constant period according to the recommendations of the lysate manufacturer (usually at 37 +1 ºC for 60 + 2 min), avoiding vibration. Test the integrity of the gel: for tubes, take each tube in turn directly from the incubator and invert it through approximately 180º in one smooth motion. If a firm gel has formed that remain in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed. The test is not valid unless the lowest concentration of the standard solutions shows a negative result in all replicate tests. The end-point is the last positive result in the series of decreasing concentrations of endotoxin. Calculate the mean value of the logarithms of the end-point concentrations and then the antilogarithm of the mean value using the following expression:

Geometric Mean end-point concentration = antilog ∑ e / ƒ

∑ e = sum of the log end-point concentrations of the dilution series used, ƒ = number of replicates. The Geometric Mean end-point concentration is the measured sensitivity of the lysate solution (IU/mL). If this is not less than 0.5λ and more than 2λ, the labeled sensitivity is confirmed and is used in the tests performed with this lysate.

Procedure: I. Preparation of Endotoxin dilutions from Control Standard Endotoxin (CSE) for control curve & for positive product control: Refer to Certificate of Analysis (COA) for matched CSE vial. Steps: 1. Reconstitute the Lyophilized material with required quantity of LRW (mentioned in the

CoA) to obtain mentioned concentration of EU/mL with the help of pyrogen free pipette.

2. Dilute CSE to 1 EU/mL using LRW and Vortex for 1 min 3. From 1 EU/mL prepare CSE of 4λ, 2λ,λ, 0.5λ,0.25λ dilutions. Where λ = Labelled

lysate sensitivity. e.g. λ = 0.125 EU/mL .Vortex each dilution for at least 1 min. Control Standard Endotoxin Dilution Scheme: Concentration = __ EU/mL as per CoA

Tube No. CSE Concentration LAL Reagent Water

Control Standard Endotoxin

I 8λ 1 EU/mL __ mL ___ mL of __ EU/mL II 4λ 0.5 EU/mL 2 mL 2 mL of 1 EU/mL III 2λ 0.25 EU/mL 2 mL 2 mL of 0.5 EU/mL IV λ 0.125 EU/mL 2 mL 2 mL of 0.25 EU/mL V ½ λ 0.0625 EU/mL 2 mL 2 mL of 0.125 EU/mL VI ¼ λ 0.03125 EU/mL 2 mL 2 mL of 0.0625 EU/mL

Dilution Scheme Labeled Claim sensitivity of Lysate (λ) is 0.125EU/mL. Following will be the dilutions one needs to prepare for Confirmation of the labeled lysate sensitivity. From the Control Standard Endotoxin vial prepare 1 EU/mL.

LAL Test procedure: Control curve Test carried out in clean depyrogenated 10 x 75 mm tubes only. Each dilution to be tested

in quadruplicate.

Tube No. Dilution LRW (µL)

CSE Dilution in µL

Total Volume

µL 1,2 Negative

Control 100 - 100

3,4,5,6 2 λ - 100 ( of 2 λ ) 100 7,8,9,10 λ - 100 ( of λ ) 100

11,12,13,14 0.5 λ - 100 ( of 0.5 λ ) 100 15,16,17,18 0.25 λ - 100 ( of 0.25λ ) 100

To each tube, add 100 µl of LAL reagent. Mix gently & incubate in Heating Block at 37ºC ± 1ºC for 60±2 min. After incubation, remove the tubes gently from the Heating Block & slowly invert through 180 º & scroll the result. + ve gel that holds its integrity when tube is inverted 180º. - ve clear or viscous liquid which flows when tube is inverted.

1EU/mL 0.5EU/mL 0.25EU/mL 0.125EU/mL 0.0625EU/mL 0.03125EU/mL

Interpretation : Correct or acceptable results:

I II III NEG Control – – – – – –

2 λ + + + + + + + + + + + + λ + + + + – – – – + + + +

0.5 λ – – – – – – – – + + + + 0.25λ – – – – – – – – – – – –

End point at λ 2 λ 1 / 2 λ Ideal Within + one twofold dilution

Void or incorrect results:

IV V VI NEG Control – – – – + +

2 λ – – – – + + + + + + + + λ – – – – + + + + + + + +

0.5 λ – – – – + + + + + + + + 0.25λ – – – – + + + + + + + +

CSE Storage (?) Dilutions (?)

Dilutions (?) Accessories LRW (?)

Geometric Mean Calculation- Example

Tube Blank 2λ λ 0.5 λ 0.25λ End point 1 – + + – – 0.125 EU/mL2 – + + + – 0.0625 EU/mL3 – + + – – 0.125 EU/mL4 – + + + – 0.0625 EU/mL

Formula:

GM end point Concentration = antilog (∑e/ƒ) Where, ∑e = Sum of log of Endpoint Concentrations ƒ = Number of Replicates

Calculation:

GM = log (0.125) + log (0.0625) + log (0.125) + log (0.0625) 4 = Anti[(-0.9030) + (-1.2041) + (-0.9030) + (-1.2041)] 4 = -4.214 4 = Antilog [(-1.0536)] = 0.0883 EU/mL

Label Sensitivity is confirmed if GM endpoint is between 2λ and ½ λ .Since 0.883EU/mL is in between 0.25EU/mL and 0.06EU/mL, Label Sensitivity is confirmed.

Inhibition / Enhancement Test and Product Validation (ii) TEST FOR INTERFERING FACTORS Prepare solutions A, B, C and D as shown in Table 2.6.14-1, and use the test solutions at a dilution less than the MVD, not containing and detectable endotoxins, operating as described under 1. Preparatory testing, (i) Confirmation of the labeled lysate sensitivity. Solution A = solution of the preparation being examined that is free of detectable endotoxins. Solution B = test for interference. Solution C = control of the labelled lysate sensitivity. Solution D = negative control (water for BET). The geometric mean end-point concentrations of solutions B and C are determined using the expression described in 1.Preparatory testing, (i) Confirmation of the labelled lysate sensitivity. The test for interfering factors is repeated when any changes are made to the experimental conditions that are likely to influence the result of the test. The test is not valid unless all replicates of solutions A and D show no reaction and the result of solution C confirms the labelled lysate sensitivity. If the sensitivity of the lysate determined with solution B is not less than 0.5λ and not greater than 2λ, the test solution does not contain interfering factors under the experimental conditions used. Otherwise, the solution interferes with the test. If the preparation being examined interferes with the test at a dilution less than the MVD, repeat the test for interfering factors using a greater dilution, not exceeding the MVD. The use of a more sensitive lysate permits a greater dilution of the preparation being examined and this may contribute to the elimination of interference. Interference may be overcome by suitable treatment, such as filtration, neutralization, dialysis or heat treatment. To establish that the treatment chosen effectively eliminates interfering factor using the preparation being examined to which the standard endotoxin has been added and which has then been submitted to the chosen treatment.

INHIBITION / ENHANCEMENT TEST Solution Endotoxin Conc. /

Solution to which

Endotoxin is added

Diluent Dilution

Factor

Initial

Endotoxin

Conc.

No. of

Replicates

A None/sample solution --- --- --- 4

B 2λ / sample solution Sample

solution

1 2λ 4

2 1λ 4

4 0.5λ 4

8 0.25λ 4

C 2λ / water for BET LAL

Reagent

Water

1 2λ 2

2 1λ 2

4 0.5λ 2

8 0.25λ 2

D None / LRW --- --- --- 2

Solution A: a sample solution of the preparation under test that is free of detectable endotoxins. Solution B: test for interference. Solution C: control for labeled LAL Reagent sensitivity. Solution D: negative control of LAL Reagent Water

SCREENING FOR INTERFERENCE: Step I 1. Calculate the MVD for your product. 2. Prepare 1/32 MVD, 1/16 MVD, ⅛ MVD, ¼ MVD, ½ MVD, dilutions of your

product. 3. Test at 1/16 MVD, ⅛ MVD, ¼ MVD, ½ MVD, MVD. 4. The lowest dilution / highest concentration at which PPC is positive is the

Non Interfering Dilution. (NID) and None Interfering Conc. (NIC). 5. Choose any dilution / conc. between NID / NIC to MVD and carry out

product validation. Test as follows – in duplicate

Tube LRW (µL)

Sample (µL)

CSE (µL)

LAL (µL)

NPC ( 1/16MVD ) 50 50 ( 1/32 MVD) – 100

PPC – 50 ( 1/32 MVD) 50(4λ) 100

NPC ( ⅛ MVD ) 50 50 ( 1/16 MVD) – 100

PPC – 50 ( 1/16 MVD) 50(4λ) 100

NPC ( ¼ MVD ) 50 50 ( ⅛ MVD ) – 100

PPC – 50 ( ⅛ MVD ) 50(4λ) 100

NPC ( ½ MVD ) 50 50 ( ¼ MVD ) – 100

PPC – 50 ( ¼ MVD ) 50(4λ) 100

NPC ( MVD ) 50 50 ( ½ MVD ) – 100

PPC – 50 ( ½ MVD ) 50(4λ) 100

The Least DILUTION / Highest CONC. of product for which PPC is Positive and NPC is Negative is the Non Interfering Dilution (NID)/ Non Interfering Concentration (NIC).

EXAMPLE

Drug : Cefotaxime Sodium Sterile 1000mg/4mL

Endotoxin Limit : NMT 0.2 EU/mg

Lysate sensitivity : 0.125 EU/mL

MVD = Potency x E.L

λ

= 250 mg/ mL X 0.2 EU/mg

0.125EU/mL

MVD = 400

If you get the following results –

Dilutions 1/16 MVD (1:25)

⅛ MVD (1:50)

¼ MVD (1:100)

½ MVD (1:200)

MVD (1:400)

Conc. in mg/mL 10 5 2.5 1.25 0.625 NPC – – – – – – – – – – PPC – – + + + + + + + +

Then the Non-Interfering Dilution is ⅛ MVD=1:50, Non-Interfering Concentration is 5 mg/mL

PRODUCT VALIDATION: Since the Non- interfering dilution is ⅛ MVD (1:50). Product can be validated between ¼ MVD and MVD. Validation at ½ MVD (1:200). Lysate sensitivity (λ) is 0.125EU/mL.

Test Sample LRW Sample (1:100) CSE LAL

Negative Water Control 100 µL − − 100 µL

2 λ(0.25EU/mL) − − 100 µL of 2 λ 100 µL

λ(0.125EU/mL) − − 100 µL of λ 100 µL

0.5 λ (0.06EU/mL) − − 100 µL of 0.5 λ 100 µL

0.25 λ (0.03EU/mL) − − 100 µL of 0.25 λ 100 µL

Negative Product Control 50 µL 50 µL − 100 µL

Positive product control 2λ − 50 µL 50 µL of 4λ 100 µL

Positive product control λ − 50 µL 50 µL of 2λ 100 µL

Positive product control 0.5 λ − 50 µL 50 µL of λ 100 µL

Positive product control 0.25 λ − 50 µL 50 µL of 0.5 λ 100 µL

RESULTS:

Endotoxin Concentration

EU/mL. Test

2λ λ 0.5 λ 0.25 λ

Negative

Control

Negative

Product

Control

Test Endpt.

EU/mL

+ + – – – 0.125

+ + – – – 0.125

+ + – – 0.125

CSE

Water

Control + + – – 0.125

+ + + – – 0.0625

+ + + – – 0.0625

+ + – – – 0.125

Positive

Product

Control + + – – – 0.125

GEOMETRIC MEAN CALCULATION GM end point Concentration = antilog ( ∑ e / ƒ ) GM CSE in Water = log (0.125) + log (0.125) + log (0.125) + log( 0.125) 4 = Anti[(-0.9030) + (-0.9030) +(-0.9030) + (-0.9030)] 4 = Antilog [(-0.9030)] = 0.125 EU/mL GM CSE in Product = log (0.0625) + log (0.0625) + log (0.125) + log( 0.125) 4 = Anti[(-1.2041)+ (-1.2041)]+(-0.9030) + (-9030)] 4 = Antilog [(-1.0536)] = 0.0883 EU/mL Since Geometric mean of CSE in water and CSE in product is between 2λ (0.25EU/mL) and ½ λ (0.0625EU/mL) product is validated at ½ MVD (1:200).

2. LIMIT TEST (METHOD A) (i) PROCEDURE Prepare solution A, B, C and D as shown in Table 2.6.14 and perform the test on these solutions following the procedure described under 1. Preparatory testing, (i) Confirmation of the labelled lysate sensitivity. Prepare solution A and solution B (positive product control) using a dilution not greater than the MVD and treatments as described in 1. Preparatory testing, (ii) Test for interfering factors. Solutions B and C (positive controls) contain the standard endotoxin at a concentration corresponding to twice the labelled lysate sensitivity. Solution D (negative control) consists of water for BET.

Solution Endotoxin Conc/ Solution to which Endotoxin is added No. of replicates

A None / Diluted sample solution 2

B 2λ / diluted sample solution 2

C 2λ / LAL Reagent Water 2

D None / LAL Reagent Water 2

(ii) Interpretation The test is not valid unless both replicates of the 2 positive control solutions B and C are positive and those of the negative control solution D are negative. The preparation being examined complies with the test when a negative result is found for both replicates of solution A. When a positive result is found for both replicates of solution A:

- if the preparation being examined is diluted to the MVD, it does not comply with the test,

- if the preparation being examined is diluted to a dilution less than the MVD, the test is repeated at a dilution not greater than the MVD.

Repeat the test if a positive result is found for one replicate of solution A and a negative result is found for the other. The preparation being examined complies with the test if a negative result is found for both replicates of solution A in the repeat test.

Example :

Drug : Cefotaxime Sodium Sterile 1000 mg/ 4mL

Endotoxin Limit : NMT 0.2 EU/mg

Lysate sensitivity : 0.125 EU/mL

MVD = Potency x E.L

λ

= 250mg/ mL X 0.2 EU/mg

0.125 EU/mL

MVD = 400

Limit Test (Routine Test) Using 50-50 Method. If you want to test sample at MVD then prepare sample at ½ MVD and CSE concentration 4λ. In above case MVD is 1:400 hence sample is prepared at ½ MVD (1:200) and CSE concentration at 4λ i e. 0.5EU/mL (λ = 0.125EU/mL).

LRW Sample (1:200)

CSE (4λ) LAL

NWC 100 µL − − 100 µL PWC 50 µL − 50 µL 100 µL NPC 50 µL 50 µL − 100 µL PPC − 50 µL 50 µL 100 µL

Limit Test (Routine Test) Using ‘HOT SPIKE’ Method.

In this method sample is directly prepared and tested at same dilution and CSE dilution is prepared at 20λ = 20 x 0.125EU/mL = 2.5EU/mL

LRW Sample (1:400)

CSE (20λ) LAL

NWC 100 µL − − 100 µL PWC 100 µL − 10 µL 100 µL NPC − 100 µL − 100 µL PPC − 100 µL 10 µL 100 µL

Expected Results

NWC – – PWC + +

Sample results

NPC – – + + – – – + PPC + + + + – – + +

Pass Fail Interference Repeat Interference related problems are addressed separately later in this manual. Please refer to the technical literature section also for a publication on Resolving LAL Test interferences and The Impact of Non- Endotoxin LAL Reactive Material on LAL analyses.

3. SEMI – QUANTITATIVE TEST (METHOD B)

(i) Procedure The test quantifies bacterial endotoxins in the test solution by titration to an end-point. Prepare solution A, B, C and D as shown Table 2.6.14.-3, and test these solutions according to the procedure described under 1. Preparatory testing, (i) confirmation of the labelled lysate sensitivity.

Solution Endotoxin concentration /

solution to which Endotoxin is

added

Diluent Dilution Factor

Initial Endotoxin

concentration

Number of replicates

A None / Test solutions

Water for BET 1 – 2

2 – 2 4 – 2 8 – 2

B 2λ / Test solution 1 2 C 2λ / Water for

BET 1 2

2 2 4 2 8 2

D None/ Water for BET

– 2

(ii) Calculation and interpretation The test is not valid unless the following 3 conditions are met:

(a) both replicates of solution D (negative control) are negative, (b) both replicates of solution B (positive product control) are positive, (c) the geometric mean end-point concentration of solution C is the range of 0.5λ,

to 2λ, To determine the endotoxin concentration of solution A, calculate the end-point concentration for each replicate series of dilutions by multiplying each end-point dilution factor by λ. The endotoxin concentration in the test solution is the geometric mean end-point concentration of the replicates (see the expression given under 1. Preparatory testing, (i) Confirmation of the labeled Lysate sensitivity). If the test is conducted with a diluted test solution, calculate the concentration of endotoxin in the original solution by multiplying the result by the dilution factor. If none of the dilutions of the test solution is positive in a valid test, record the endotoxin concentration as less than λ (or if a diluted sample was tested as less than λ x the lowest dilution factor of the sample). If all dilutions are positive, the endotoxin concentration is recorded as equal to or greater than the greatest dilution factor multiplied by λ . The preparation meets the requirements of the test if the endotoxin concentration is less than that specified in the individual monograph.

Example

Drug : Cefotaxime Sodium Sterile 1000 mg/ 4mL

Endotoxin Limit : NMT 0.2 EU/mg

Lysate sensitivity : 0.125 EU/mL

MVD = Potency x E.L

λ

= 250mg/ mL X 0.2 EU/mg

0.125 EU/mL

MVD = 400

1/16MVD

(1:25) ⅛MVD (1:50)

¼ MVD (1:100)

½ MVD (1:200)

MVD (1:400)

NPC – – + + – – – – – – PPC – – + + + + + + + + λ is Lysate sensitivity = 0.125EU/mL Then Endotoxin content is = Dilution factor of maximum dilution at which

NPC is positive x λ = 50 x 0.125EU/mL = 6.25 EU/mL 250 mg/mL

= 0.025 EU/mg

REFERENCE STANDARD ENDOTOXIN

USP Endotoxin Reference Standard (RSE) The variability of early endotoxin standard led to recognition that a reference standard endotoxin (RSE) was needed to confirm the sensitivity of LAL reagents. The search for a standard was complicated because endotoxin potency varied with method of purification, bacterial origin, and formulation. Rudbach and associates (1976) were engaged by the USP and the FDA to resolve this issue. The objective was selection and preparation of a stable reference endotoxin that was free of biologically active proteins, had average endotoxic activity, and could be chemically characterized. A 30g batch of reference LPS that met these criteria was prepared from Escherichia coli 0113:H10:K(-) (Rudbach, Akiya and Elin 1976). The FDA specified a means for standardizing other purified endotoxins against this reference standard. An international World Health Organization (WHO) reference endotoxin was prepared under the direction of people to harmonize the ELs so that 1 international unit (IU) of endotoxin equal 1 endotoxin unit (EU) (Poole, Dawson and Gaines Das 1997). The FDA has a sublot of the WHO reference endotoxin, known in the FDA as EC-6, which LAL suppliers use for sensitivity assays (determination of lambda,λ) of their LAL reagents. USP Endotoxin Lot G contains 10000 Units (EU) per vial. This is commercially available Functions of Reference Standard Endotoxin The RSE has three critical functions. Through its licensing process, the FDA requires all LAL producers to determine the sensitivity of LAL by an assay of a two-fold dilution series of RSE made from 1 EU/mL. Second, the RSE is the primary standard for standardizing the potency of control standard endotoxin (CSE) with a specific lot of LAL, as required by the LAL-Test Guideline (FDA 1987). Finally, RSE is used when a referee test is needed to resolve a disagreement over test results.

RSE preparation to determine the sensitivity of LAL:

Method

RSE Concentrations. EU/mL

End point

Geometric Mean

G.M. = antilog ( Σ e/ f)

Tubes Replicates 0.25 0.125 0.0625 0.03125

1 + + – – 0.125 -0.9030 2 + + – – 0.125 -0.9030 3 + + – – 0.125 -0.9030 4 + + – – 0.125 -0.9030

G.M. = antilog ( Σ (-0.9030) + (-0.9030) + (-0.9030) + ( -0.9030) /4 G.M. = antilog ( Σ -3.612/ 4) G.M. = antilog ( -0.9030) = 0.125 EU/mL

Hence Label Claim Sensitivity of this batch of lysate is 0.125 EU/mL.

Sensitivity is denoted by λ. ( Lambda )

CONTROL STANDARD ENDOTOXIN (CSE)

Purpose of Control Standard Endotoxin Because the RSE is expensive and exhaustible, LAL reagent suppliers produce CSE for routine tests. The sole purpose of CSE is to serve as a surrogate for RSE during routine endotoxin testing. The roles of CSE are to (1) confirm LAL test validity by recovery of positive product controls (PPC) and (2) verify control of test parameters (e.g., reagents, accessories, analyst proficiency) through recovery of label-claim sensitivity (λ) during routine BET assays. In the Pharmacopoeial Forum (USP 2000), the USP stated its recognition of equivalence for RSE and CSE preparations .The USP does not mention the use of CSE and only refers to the RSE. The Preamble, Bacterial Endotoxins Test, Pharmacopoeial Forum 26: 218 – 19, 2000 allows “The use of in-house standards shown to be equivalent to USP Reference Standards is permitted under the requirements for alternative methods in the General Notices.”

CSE is the in-house standard. The Standard Testing Procedure and Standard Operating Procedure is in compliance if it requires. 1. Acceptance of CoA from the CSE vendor 2. Completion of a label claim verification. If need for USP reference to use CSE then one can refer to Pg. 1696 of USP 23 which gives the most recent description of RSE / CSE ratio determination for Gel Clot Methods. USP requires ALL CSE to have a ratio between 2 EU/ng and 50 EU/ng. Every Manufacturer of LAL provides a Control Standard Endotoxin with a certificate of analysis that contains the RSE to CSE ratio for specific LAL lot. All RSE to CSE ratios are Lot specific. Hence before using LAL one should check whether they are using the correct combination of LAL and CSE which is mentioned on the CoA.

Preparation of CoA for RSE/CSE: USP requires ALL Control Standard Endotoxins (CSE) to have a ratio between 2 EU/ng and 50 EU/ng. Variations in RSE/ CSE ratio is due to the INHERENT + 2 FOLD VARIATION IN THE LAL SENSITIVITY. Hence with different batches of LAL of same sensitivity with same lot of CSE you will have different RSE /CSE ratios. We can get different results which will result in different RSE/CSE ratios. Some examples of the results and their calculations are listed below Each CSE contains 100ng/vial. Each is assayed as follows. From each vial 0.025, 0.0125, 0.00625, 0.003125ng/mL is prepared and 0.1mL LAL is added to each tube. Assay is performed in quadruplicate. RSE to CSE ratio

RSE Concs. EU/mL

End point

Geometric Mean

G.M. = antilog ( Σ e/ f)

Tubes

0.25 0.125 0.0625 0.03125 1 + + – – 0.125 -0.9030 2 + + – – 0.125 -0.9030 3 + + – – 0.125 -0.9030 4 + + – – 0.125 -0.9030

G.M. = antilog ( Σ -0.9030 +-0.9030 +-0.9030 + -0.9030) /4 G.M. = antilog ( Σ -3.612/ 4) G.M. = antilog ( -0.9030) = 0.125 EU/mL

CSE contains 100ng /vial and is diluted to the below mentioned concentrations.

CSE Concs. ng/mL

End point

Geometric Mean

G.M. = antilog ( Σ e/ f)

Tubes

0.025 0.0125 0.00625 0.003125 1 + + – – 0.0125 -1.9030 2 + + – – 0.0125 -1.9030 3 + + – – 0.0125 -1.9030 4 + + – – 0.0125 -1.9030

G.M. = antilog ( Σ -1.9030 + -1.9030 + -1.9030 + -1.9030) /4 G.M. = antilog ( Σ -7.612/ 4) G.M. = antilog ( -0.9030) = 0.0125 ng/mL

RSE/CSE Ratio = Geometric Mean End point of RSE concentration Geometric Mean End point of CSE concentration = 0.125 EU/mL 0.0125 ng/mL = 10 EU/ng

Result 2 CSE Concentrations

Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point

1 + + + − 0.00625 2 + + − − 0.0125 3 + + − − 0.0125 4 + + − − 0.0125

Geometric Mean G.M.= Antilog (Σe/f) where e = log of end points f = No.of replicates. G.M. = Antilog [log (0.00625) + log (0.0125) + log (0.0125) + log(0.0125)] 4 = Antilog [(-2.2041) + (-1.9030) + (- 1.9030) + (-1.9030) ] 4 = Antilog [- 7.91312] 4 = Antilog [- 1.978] = 0.0105 ng/mL. RSE/CSE = 0.125 EU/mL = 11.9 = 12 EU/ng. 0.0105ng/mL

Result 3 CSE Concentrations Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point

1 + + + − 0.00625 2 + + + − 0.00625 3 + + − − 0.0125 4 + + − − 0.0125

Geometric Mean G.M.= Antilog (Σe/f) where e = log of end points f = No.of replicates. G.M. = log [ log (0.00625) + log (0.00625) + log (0.0125) + log(0.0125) ] 4 = Antilog [ (-2.2041) + (-2.2041) + (- 1.9030) + (-1.9030) ] 4 = Antilog [ - 8.2142 ] 4 = Antilog [ - 2.05355 ] = 0.0088 ng/mL. RSE/CSE = 0.125 EU/mL = 15 EU/ng. 0.008 ng/mL

Result 4 CSE Concentrations

Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point 1 + + + − 0.00625 2 + + + − 0.00625 3 + + + − 0.00625 4 + + − − 0.0125

Geometric Mean G.M.= Antilog (Σe/f) where e = log of end points f = No.of replicates. G.M. = Antilog [ log (0.00625) + log (0.00625) + log (0.00625) + log (0.0125) ] 4 = Antilog [ (-2.2041) + (-2.2041) + (-2.2041) + (-1.9030) ] 4 = Antilog [ - 8.515 ] 4 = Antilog [ - 2.128 ] = 0.0074 ng/mL. RSE/CSE = 0.125 EU/mL = 17 EU/ng. 0.007 ng/mL

Result 5 CSE Concentrations

Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point 1 + − − − 0.025 2 + − − − 0.025 3 + − − − 0.025 4 + − − − 0.025

Geometric Mean G.M.= Antilog (Σe/f) where e = log of end points f = No.of replicates. G.M. = Antilog [log (0.025) +log (0.025)+ log (0.025+ log (0.025) 4 = Antilog [(-1.6020) + (-1.6020) + (-1.6020) + (-1.6020) ] 4 = Antilog [- 6.4080 ] 4 = Antilog [- 1.6020] = 0.025 ng/mL. RSE/CSE = 0.125 EU/mL = 5EU/ng.

0.025 ng/mL Hence the CoA contains one of these ratios as calculated above.

Reconstitution of LAL Reagent LIMUSATE using BET WATER Reconstitute LAL after preparing and adding sample, CSE to the test tubes. Steps : 1. Remove aluminum seal of the Lysate vial without opening rubber stopper. 2. Carefully remove the stopper. Keep the stopper in a clean surface without touching the

inner portion of stopper. 3. Rehydrate the lyophilized powder using required quantity of LRW with the help of

depyrogenated pipette. With 1 mL LRW (for Limusate-1mL 10 test vial) With 5 mL LRW (for Limusate-5 mL 50 test vial) 4. Stopper or use parafilm to seal the reconstituted vial. Do not shake. Mix gently without formation of any air bubbles. AVOID TOUCHING OF LYSATE TO THE STOPPER. DO NOT VORTEX. Storage: Store the reconstituted LAL reagent vial at 2 to 8° C for 8 hours. Reconstituted Lysate should be preferably stored in the freezer below 0°C when it is to be used intermittently. Freeze it & use on the next day. If not, can be frozen below - 20°C.up to 2 weeks, to be frozen & thawed only once.

TESTING OF WATER FOR INJECTION USING HOT SPIKE

METHOD AND 50-50 METHOD.

Endotoxin Limit : 0.25 EU/mL LAL sensitivity : 0.125 MVD = 2 Testing at 0.125EU/mL using Hot spike.

LRW WFI CSE (20λ)

LAL

NWC 100 µL − − 100 µL PWC 100 µL − 10 µL 100 µL NPC − 100 µL − 100 µL PPC − 100 µL 10 µL 100 µL

Testing at 0.25EU/mL using 50-50 method

LRW WFI CSE (4λ)

LAL

NWC 100 µL − − 100 µL PWC 100 µL − 50 µL 100 µL NPC 50 µL 50 µL − 100 µL PPC − 50 µL 50 µL 100 µL

QUANTIFYING ENDOTOXIN IN WATER

TESTING OF WATER FOR INJECTION FOR DIFFERENT LEVELS OF ENDOTOXIN USING HOT SPIKE METHOD USING LAL λ =0.03 EU/mL.

Test directly at 0.03 EU/mL

LRW WFI CSE (20λ) LAL NWC 100 µL − − 100 µL PWC 100 µL − 10 µL 100 µL NPC − 100 µL − 100 µL PPC − 100 µL 10 µL 100 µL

If NPC −ve, PPC +ve report WFI contains less than 0.03 EU/mL. If NPC +ve, PPC +ve report WFI contains 0.03 EU/mL Prepare 1:2 and test at 0.06 EU/mL

LRW WFI CSE (20λ) LAL NWC 100 µL − − 100 µL PWC 100 µL − 10 µL 100 µL NPC − 100 µL − 100 µL PPC − 100 µL 10 µL 100 µL

If NPC −ve, PPC +ve report WFI contains less than 0.06 EU/mL If NPC +ve, PPC +ve report WFI contains 0.06 EU/mL Prepare 1:4 and test at 0.125 EU/mL

LRW WFI CSE (20λ) LAL NWC 100 µL − − 100 µL PWC 100 µL − 10 µL 100 µL NPC − 100 µL − 100 µL PPC − 100 µL 10 µL 100 µL

If NPC −ve, PPC +ve report WFI contains less than 0.125 EU/mL If NPC +ve, PPC +ve report WFI contains 0.125 EU/mL Prepare 1:8 and test at 0.25 EU/mL

LRW WFI CSE (20λ) LAL NWC 100 µL − − 100 µL PWC 100 µL − 10 µL 100 µL NPC − 100 µL − 100 µL PPC − 100 µL 10 µL 100 µL

If NPC −ve, PPC +ve report WFI contains less than 0.25 EU/mL If NPC +ve, PPC +ve report WFI contains 0.25 EU/mL and report failure.

NUCLEAR MEDICINE

Product : Fludeoxyglucose F18 Injection

EL : 25 EU/mL

LAL sensitivity : 0.03EU/mL (for all calculations sensitivity = 0.03125EU/mL)

MVD : 800.

Prepare 1:800 dilution & test as follows.

From CSE = 20EU/mL, prepare 20 λ = 20 x 0.03125 = 0.625EU/mL

Tube no.

Concentration LRW(µL) Sample(µL) CSE(µL) LAL (µL)

Reading

1,2,3 NPC - 100(1:800) -- 100 1 tube at 20 mins & 2 tubes at 60 mins.

4,5 PPC-2λ - 100(1:800) 10µL of 20λ 100 Both tubes at 60 mins

6 PPC-160λ - 100(1:800) 25µL (20EU/mL) 100 At 20 mins. 7,8 PWC - - 100(2λ) 100 At 60 mins. 9,10 NWC 100 - - 100 At 60 mins.

DRY HEAT STERILISATION The USP requires you to show a 3 log reduction in Endotoxin The Validation procedure here assumes that a loaded oven in the maximum configuration would serve as a ‘worst case’ model. Time and Temperature Profile

Depends upon ovens/tunnels specifications and type As temperature is increased, time required is less for approximately the same amount

of thermal destruction 250°C for 1 hour for DHS 350°C for 4-6minutes for Tunnel Revalidation if profile is changed

Temperature Profile Unloaded Vs. Loaded Oven

Check proper installation Operator’s manual Utility connections Structural dimensions Accessories like heater, blowers Thermocouple study, 10 probes/100cu.ft. Thermocouple study, loaded.

As per the USP 29, Chapter 1211 defines an acceptable cycle for depyrogenation as one which gives a three log reduction in the endotoxin concentration at the end of the cycle. Endotoxin Challenge of the Loaded Oven

Endotoxin Challenge vials / Indicators containing 10000ng approx =100000EU/vial or 1000ng approx = 10000EU/vial

Minimum 1000EU/vail concentration Quantify more than 3 log reduction Minimum Lysate sensitivity Not Less Than 0.15EU/mL .Use LAL of sensitivity

0.125EU/mL,0.06EU/mL,0.03EU/mL.

VALIDATION PROTOCOL FOR DRY HEAT DEPYROGENATION CYCLE

PREPARATION 1. Remove seals. 2. Aseptically remove stoppers and discard. 3. Cover endotoxin vials with a double layer of aluminum foil. HEAT EXPOSURE OF ENDOTOXIN INDICATORS 1. All endotoxin indicators shall be labeled by a marking pen as to oven location. 2. Position one endotoxin indicator adjacent to each thermocouple. 3. Two vials per depyrogenation cycle shall be retained as a positive control. These controls

should be placed outside the dry heat oven. 4. Expose indicators to heat cycle. 5. After oven run is complete, carefully remove indicator vials. ENDOTOXIN RECOVERY ASSAY 1. Initiate recovery by rehydrating the endotoxin indicator vials including positive controls

with 1mL of LRW. Immediately vortex each container for 5 minutes. 2. Controls: a. Positive Endotoxin Indicator Controls Make 10-fold and 2-fold serial dilutions to get the concentrations (8λ,4λ,2λ,λ,λ/2,λ/4)

This is the basis for determining the endotoxin content. Actual recovery concentration is to be taken for calculation.

b. Standard Curve Make a valid 4-point standard curve of the CSE using concentrations 2λ,λ,λ/2,λ/4 where

λ is the sensitivity of the Lysate or use a Lysate whose sensitivity is verified. c. Negative Control The LRW used above is the negative control.

CALCULATIONS 1. Recovered EU/mL from heat = Reciprocal of last dilution x λ treated Sample of sample that was positive 2. Recovered EU/mL from Positive = Reciprocal of last dilution x λ Endotoxin Indicator Controls of PC that was positive (PC) 3. Log {Recovered EU/mL from PC} - Log {Recovered EU/mL from Sample} = X - Log Reduction INTERPRETATION OF RESULTS 1. The depyrogenation cycle was successful if there was a > 3-log reduction in endotoxin in

the heat exposed vials.

FLOW CHART FOR VALIDATION OF DRY HEAT STERILIZERS OVENS / TUNNELS

Start with a 10000ng approx =100,000 EU Endotoxin Indicator vial. ↓

Reconstitute the vial in 2mL of LRW, Vortex for 20 minutes. ↓

Aliquot 0.1 mL into ampoules or vials used for your injectables. The vials / ampoules must be the same ones used typically for that particular oven / tunnel.

↓ Dry the vials / ampoules in a Laminar Flow hood overnight. Each vial / ampoule will

now contain approx 5,000 EU. ↓

Keep at least 1 vial / amp as Positive Control (do not pass through oven / tunnel). Mark as PPC-1, 2, etc.

↓ Expose the rest (8 or 9 amps / vials to appropriate number) to the oven / tunnel

as per it’s typical depyrogenation cycle. ↓

After the cycle, mark all the vials as NPC - 1,2,3 etc. ↓

Reconstitute all PPC and NPC vials / amps in 1mL of LRW each. Vortex each vial / amp for at least 5 minutes. Remember to be consistent.

↓ For PPC vial / amp For NPC vials / amps (approx 5,000 EU/mL) (Unknown - Assuming a 3 log reduction to 5 EU/mL) ↓ 1:50 dilution ↓ 1:10 dilution 100 EU/mL 0.5 EU/mL ↓ 1:100 dilution ↓ 1:4 dilution 1 EU/mL 0.125 EU/mL (do 2 - fold dilutions to run a standard dilution series) 8λ,4λ,2λ ,λ,λ / 2,λ / 4 Test all final dilutions in DUPLICATE with a LAL reagent of 0.125EU/mL sensitivity. Results : For a valid Depyrogenation cycle, a greater than 3-log reduction of Endotoxin is to be achieved.

FLOW CHART FOR VALIDATION OF DRY HEAT STERILIZERS OVENS / TUNNELS

Start with a 10000ng approx =100,000 EU Endotoxin Indicator vial. ↓

Reconstitute the vial in 2 mL or 1mL of LRW, Vortex for 20 minutes. ↓

Aliquot 0.2 mL or 0.1mL (depending on volume of reconstitution of the Endotoxin Indicator vial that is 2mL or 1mL) into ampoules or vials used for your injectables. The vials /

ampoules must be the same ones used typically for that particular oven / tunnel. ↓

Dry the vials / ampoules in a Laminar Flow hood overnight. Each vial / ampoule will now contain approx 10,000 EU.

↓ Keep at least 1 vial / amp as Positive Control (do not pass through oven / tunnel).

Mark as PPC-1, 2, etc. ↓

Expose the rest (8 or 9 amps / vials to appropriate number) to the oven / tunnel as per it’s typical depyrogenation cycle.

↓ After the cycle, mark all the vials as NPC - 1,2,3 etc.

↓ Reconstitute all PPC and NPC vials / amps in 1mL of LRW each. Vortex each

vial / amp for at least 5 minutes. Remember to be consistent. ↓

For PPC vial / amp For NPC vials / amps (approx 10,000 EU/mL) (Unknown - Assuming a 3 log reduction to 10 EU/mL) ↓ 1:10 dilution ↓ 1:10 dilution 1000 EU/mL 1 EU/mL ↓ 1:10 dilution ↓ 1:8 dilution 100 EU/mL 0.125 EU/mL ↓ 1:10 dilution 10 EU/mL ↓ 1:10 dilution 1 EU/mL (do 2 - fold dilutions to run a standard dilution series) 8λ,4λ,2λ ,λ,λ / 2,λ / 4 Test all final dilutions in DUPLICATE with a LAL reagent of 0.125 EU/mL sensitivity. Results: For a valid Depyrogenation cycle, a greater than 3-log reduction of Endotoxin is to be achieved.

Locations of Vials in DHS

TOP

FRONT

1 2

3

4

5

6 7

8

9

Locations of Vials in Tunnel

1

3

2 4

5

6

7

8

9

Direction of conveyer belt

Examples of Product Validation

Preliminary Screening Test

Product : Gentamycin Inj. Potency : 40 mg/mL Endotoxin Limit : 0.71EU/mg Lysate Sensitivity (λ): 0.125EU/mL MVD : 1: 227 Test Dilution: 1:25, 1:50, 1:100, 1:200 Test Concentrations: 1.6 mg/mL, 0.8 mg/mL, 0.4 mg/mL, 0.2 mg/mL Test Dilutions / Concentrations NPC PPC

1:25 − − + + 1:50 − − + + 1:100 − − + + 1:200 − − + +

++ : Gel Formation − − : No Gel Formation

Conclusion: No inhibition observed. Test dilution of 1:100 to be chosen for validating the product.

LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION AS AN END-PRODUCT ENDOTOXINS TEST

Product : Gentamycin Inj Preparation : Product Concentration : 40 mg/mL Endotoxin Limit : NMT 0.71 EU/mg. MVD: 1:227 Test Concentration / Dilution: 0.4 mg/mL; 1:100

Sample Preparation : 1:50

Results:

Test Material

Endotoxin Concentration EU/mL.

NEG Control

NEG Product Control

Test Endpt. EU/mL

Log of Endpt

Geometric Mean

2λ λ 0.5λ 0.25λ CSE + + − − − 0.125 -0.903

Water + + − − − 0.125 -0.903 Control + + − − 0.125 -0.903

+ + − − 0.125 -0.903

0.125 EU/mL

Positive + + + − − 0.0625 -1.2041 Product + + + − − 0.0625 -1.2041 Control + + + − − 0.0625 -1.2041

+ + + − − 0.0625 -1.2041

0.0625 EU/mL

+ = Firm Gel − = No Gel or less than firm Gel Interpretation: Test results are VALID Comments : The end points of CSE in Water = 0.125 EU/mL. The end point of CSE in Product = 0.0625 EU/mL which is within ± one two fold dilution of the LAL labeled sensitivity λ. λ = 0.125 EU/mL.

Preliminary Screening Test Product : Dextrose for inj. (5% W/V). Endotoxin Limit : 0.5 EU/mL Lysate Sensitivity (λ) : 0.06EU/mL MVD : 1:8 Test Dilution : 1:2, 1:4, 1:8 Test Dilutions / Concentrations NPC PPC

1:2 − − + + 1:4 − − + + 1:8 − − + +

++ : Gel Formation − − : No Gel Formation

Conclusion: No inhibition observed. Test dilution of 1:4 to be chosen for validating the

product.

LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION

AS AN END-PRODUCT ENDOTOXINS TEST

Product : Dextrose for inj. (5% W/V). Preparation : Endotoxin Limit : NMT 0.5EU/mL. MVD: 1:8 Test Dilution: 1:4 Sample Preparation: 1:2 Results:

Test Material

Endotoxin Concentration EU/mL.

NEG Control

NEG Product Control

Test Endpt. EU/mL

Log of Endpt.

Geometric Mean

2λ λ ½λ ¼λ CSE + + − − − 0.0625 -1.2041

Water + + − − − 0.0625 -1.2041 Control + + − − 0.0625 -1.2041

+ + − − 0.0625 -1.2041

0.0625 EU/mL

Positive + + − − − 0.0625 -1.2041 Product + + − − − 0.0625 -1.2041 Control + + − − − 0.0625 -1.2041

+ + − − − 0.0625 -1.2041

0.0625 EU/mL

+ = Firm Gel − = No Gel or less than firm Gel Interpretation: Test results are VALID. Comments : The end points of CSE in Water = 0.0625 EU/mL. The end point of CSE in Product = 0.0625 EU/mL which is within ± one two fold dilution of the LAL labeled sensitivity λ. λ = 0.06 EU/mL.

Preliminary Screening Test Product : Normal Saline Endotoxin Limit : 0.5 EU/mL Lysate Sensitivity (λ) : 0.06EU/mL MVD : 1:8 Test Dilution : 1:2, 1:4, 1:8 Test Dilutions / Concentrations NPC PPC

1:2 − − + + 1:4 − − + + 1:8 − − + +

++ : Gel Formation − − : No Gel Formation

Conclusion: No inhibition observed. Test dilution of 1:4 to be chosen for validating the product.

LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION AS AN END-PRODUCT ENDOTOXINS TEST

Product : Normal Saline Preparation : Endotoxin Limit : NMT 0.5EU/mL. MVD : 1:8 Test Dilution : 1:4 Sample Preparation : 1:2 Results :

Test Endotoxin Concentration EU/mL.

NEG Control

NEG Product Control

Test Endpt. EU/mL

Log of Endpt.

Geometric Mean

Material 2λ λ 0.5λ 0.25λ CSE + + − − − 0.0625 -1.2041

Water + + − − − 0.0625 -1.2041 Control + + − − 0.0625 -1.2041

+ + − − 0.0625 -1.2041

0.0625 EU/mL

Positive + + + − − 0.03125 -1.5051 Product + + + − − 0.03125 -1.5051 Control + + + − − 0.03125 -1.5051

+ + + − − 0.03125 -1.5051

0.03125 EU/mL

+ = Firm Gel − = No Gel or less than firm Gel Interpretation: Test results are VALID Comments : The end points of CSE in Water = 0.0625 EU/mL. The end point of CSE in Product = 0.03125 EU/mL which is within ± one two fold dilution of the LAL labeled sensitivity λ. λ = 0.0625 EU/mL.

Preliminary Screening Test Product : Benzathine Penicillin. Potency : 400,000 units/mL Endotoxin Limit : 0.01 EU/100 units Lysate Sensity (λ) : 0.125EU/mL MVD : 1:320 Test Dilution: 1:40, 1:80, 1:160, 1:320 Test Concentrations: 1.6 mg/mL, 0.8 mg/mL, 0.4 mg/mL, 0.2 mg/mL Test Dilutions / Concentrations NPC PPC

1:25 − − + + 1:50 − − + + 1:100 − − + + 1:200 − − + +

++ : Gel Formation − − : No Gel Formation

Conclusion : No inhibition observed. Test dilution of 1:100 to be chosen for validating the product.

LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION AS AN END-PRODUCT ENDOTOXINS TEST

Product : Gentamycin Inj Preparation : Product Concentration: 40mg/mL Endotoxin Limit : NMT 0.71 EU/mg. MVD: 1:227 Test Concentration / Dilution: 0.4 mg/mL; 1:100 Sample Preparation: 1:50 Results :

Test Material

Endotoxin Concentration EU/mL.

NEG Control

NEG Product Control

Test Endpt. EU/mL

Log of Endpt.

Geometric Mean

2λ λ ½λ ¼λ CSE + + − − − 0.125 -0.903

Water + + − − − 0.125 -0.903 Control + + − − 0.125 -0.903

+ + − − 0.125 -0.903

0.125 EU/mL

Positive + + + − − 0.0625 -1.2041 Product + + + − − 0.0625 -1.2041 Control + + + − − 0.0625 -1.2041

+ + + − − 0.0625 -1.2041

0.0625 EU/mL

+ = Firm Gel − = No Gel or less than firm Gel Interpretation : Test results are : VALID INVALID Comments : The end points of CSE in Water = 0.125 EU/mL. The end point of CSE in Product = 0.06 EU/mL which is within ± one two fold dilution of the LAL labeled sensitivity λ. λ = 0.125 EU/mL