An LAL Timeline

Note that many of the referenced materials can be found in our Horseseshoe Crab Research Database.

1885

First recorded scientific observation of the coagulation of Limulus’ blood.

Observations upon the chemical composition and coagulation of the blood of Limulus polyphemus, Callinectes hastatus, and Cucumaria sp. Johns Hopkins Univ Circ 5:4.

1953

Frederik B. Bang describes the effects of injecting a marine bacterium into Limulus polyphemus. His results indicate this causes intravascular clotting; other Gram-negative bacteria could cause similar results; the clotting did not require a living bacteria, the bacterial component that caused clotting was not heat labile; and, Gram-positive bacteria did not produce this effect. This finding was the foundation which ultimately lead to the discovery of LAL many years later.

Bang, FB (1953) The toxic effect of a marine bacterium on Limulus and the formation of blood clots. Biol Bull 105:447-448.

1956

Renewed interest in Limulus polyphemus as a biological model for the study of disease mechanisms.

Bang, FB (1956) A bacterial disease of Limulus polyphemus. Bull Johns Hopkins Hosp 98:325.

1964

First detailed modern description of cellular coagulation in Limulus.

Levin, J, and Bang, FB (1964) A description of cellular coagulation in Limulus. Bull Johns Hopkins Hosp 115:337.

1964

Discovery that endotoxin is the key factor in clotting of Limulus blood.

Levin, J, and Bang, FB, (1964) The role of endotoxin in the extracellular coagulation of Limulus blood. Bull Johns Hopkins Hosp 115:265.6.

1968

Discovery that bacterial endotoxin (pyrogen) was responsible for the clotting of Limulus blood and that the mechanism was located in the amebocyte granules.

Levin, J. Bang, FB (1968) Clottable protein in Limulus: Its localization and kinetics of its coagulation by endotoxin. Thromb Diathes Haemorrh (Stuttg) 19:186.

1969

James F. Cooper begins a study under the direction of Jack Levin and Henry N. Wagner to explore the use of LAL as an alternative to using the rabbit pyrogen test to detect endotoxin in pharmaceuticals.

 

1970

First application of LAL to the diagnosis of human disease.

Levin, J, Tomasulo, PA, and Oser, RS (1970) Detection of endotoxin in human blood and demonstration of an inhibitor. J Lab Clin Med 75:903.

1971

LAL shown to correlate well with other assays for endotoxin, e.g. Pyrogen (rabbit) Test.

Cooper, JF, Levin, J, and Wagner, HN Jr. (1971) Quantitative comparison of in vitro and in vivo methods for the detection of endotoxin. J Lab Clin Med 78(1):138.

1972

LAL shown it could be applied to the detection of endotoxin in pharmaceutical drugs.

Cooper, JF, Hochstein, HD, Seligmann, EB Jr. (1972) The Limulus test for endotoxin (pyrogen) in radiopharmaceuticals and biologicals. Bull Parenter Drug Assoc 26(4):153.

1973

Food and Drug Administration first proposes guidelines for the manufacture of LAL.

Federal Register January 1973. Federal Register Vol. 38, No. 8 p. 1404.

1973

Food and Drug Administration proposes standards for the manufacture of LAL. September 18, 1973.

United States Federal Register Vol. 38, No.180. Limulus Amebocyte Lysate: Additional standards. p. 26103-26132.

1973

Cooper, Hochstein, and Seligmann Drafted “Return To Sea” Policy.

 

1974

Travenol Laboratories, Inc. establishes a lysate production laboratory at their Kingstree, South Carolina plant and is using their LAL to test pharmaceuticals both domestically and in some international plants by 1974.

Mascoli, Carmine C, and Marlys E. Weary. (1979) Applications and advantages of the Limulus amebocyte lysate (LAL) pyrogen test for parenteral injectable products. In Progress in Clinical and Biological Research, Vol. 29, Biomedical Applications of the Horseshoe Crab (Limulidae), Elias Cohen, et. al. eds. Alan R. Liss, Inc., New York (Proceedings of a Symposium Held at the Marine Biological Laboratory, Woods Hole, Massachusetts, October, 1978)

1977

Beginning with Associates of Cape Cod, Inc., Woods Hole, MA, in September 1977, by October 1978 there were three additional LAL manufacturers licensed by the FDA. These were, in order of licensing: Mallinckrodt Inc., St. Louis, MO, Microbiological Associates, Walkersville, MD, and Travenol Laboratories, Morton Grove, IL

 

1977

FDA allows substitution of LAL for the official rabbit pyrogen test when testing biological products and medical devices providing approval is first obtained from the appropriate bureau of the FDA.

Federal Register, November 4, 1977, Vol. 42. No. 213, p 57749.

1978

FDA proposal for the live release of horseshoe crabs back to their native environment after only one blood collection.  (This regulation was rescinded in 1996 under the Federal Reinvention of Government (REGO) Initiative.)

United States Federal Register (1978) 43:35731-35734. 

1979

Attempt at Limulus aquaculture.

Kropach, Chaim (1979) Observations on the potential of Limulus aquaculture in Israel. In Progress in Clinical and Biological Research, Vol. 29, Biomedical Applications of the Horseshoe Crab (Limulidae), Elias Cohen, et. al. eds. Alan R. Liss, Inc., New York (Proceedings of a Symposium Held at the Marine Biological Laboratory, Woods Hole, Massachusetts, October, 1978)

1979

Worthington Laboratories, Freehold, NJ joins the group of laboratories manufacturing LAL (PyrostatTM brand).

Teller, Joseph D., and Kristine M. Kelly. (1979) A turbidimetric Limulus amebocyte lysate assay for the quantitative determination of gram negative bacterial endotoxin. In Progress in Clinical and Biological Research, Vol. 29, Biomedical Applications of the Horseshoe Crab (Limulidae), Elias Cohen, et. al. eds. Alan R. Liss, Inc., New York (Proceedings of a Symposium Held at the Marine Biological Laboratory, Woods Hole, Massachusetts, October, 1978)

1979

Warning that: “The emerging importance of Limulus to biomedical research, as the source of Limulus lysate, requires more complete knowledge of the biology of the species, so that it can be wisely managed as a natural resource.”

Rudloe, Ann (1979) Limulus polyphemus: A review of the ecologically significant literature. In Progress in Clinical and Biological Research, Vol. 29, Biomedical Applications of the Horseshoe Crab (Limulidae), Elias Cohen, et. al. eds. Alan R. Liss, Inc., New York (Proceedings of a Symposium Held at the Marine Biological Laboratory, Woods Hole, Massachusetts, October, 1978)

1979

Warning that biomedical use as well as use of Limulus for conch bait in addition to habitat destruction requires preparation of a practical plan for the conservation of Limulus.

Galler, Sidney R. (1979) Limulus polyphemus, a target of opportunity. In Progress in Clinical and Biological Research, Vol. 29, Biomedical Applications of the Horseshoe Crab (Limulidae), Elias Cohen, et. al. eds. Alan R. Liss, Inc., New York (Proceedings of a Symposium Held at the Marine Biological Laboratory, Woods Hole, Massachusetts, October, 1978)

1979

Attempt to grow Limulus’ amebocytes in culture (for the in vitro production of LAL).

Pearson, Frederick C, and Ellen Woodland (1979) The In Vitro cultivation of Limulus amebocytes. In Progress in Clinical and Biological Research, Vol. 29, Biomedical Applications of the Horseshoe Crab (Limulidae), Elias Cohen, et. al. eds. Alan R. Liss, Inc., New York (Proceedings of a Symposium Held at the Marine Biological Laboratory, Woods Hole, Massachusetts, October, 1978).

1979

Review of Limulus and other invertebrate clotting with emphasis on innate infection defense mechanisms.

Bang, Frederik B (1979) Ontogeny and phylogeny of response to Gram-negative endotoxin among the marine invertebrates. In Progress in Clinical and Biological Research, Vol. 29, Biomedical Applications of the Horseshoe Crab (Limulidae), Elias Cohen, et. al. eds. Alan R. Liss, Inc., New York (Proceedings of a Symposium Held at the Marine Biological Laboratory, Woods Hole, Massachusetts, October, 1978)

1980

In the FEDERAL REGISTER of January 18, 1980 (45 FR 3668), FDA announced the availability of a draft guideline that set forth procedures for use of the LAL test as an end-product testing method for endotoxins in human and animal injectable drug products. This draft guideline was made available to interested parties to permit manufacturers, especially those who had used the LAL test in parallel with the rabbit pyrogen test, to submit data that could be considered in the preparation of any final guideline.

 

1980

The United States Pharmacopeial Convention (USP) publishes General Chapter <85> Bacterial Endotoxins Test in USP XX, making the LAL test a Compendial method to detect bacterial endotoxin in pharmaceutical products and medical devices.

 

1987

The United States Food and Drug Administration publishes Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices, describing FDA’s opinion regarding the appropriate methods for validation and use of LAL for detecting the presence of endotoxin in medical products.

 

1991

Dr. James and Frances Cooper wrote State legislation for approval in 1991 that provided for the management and regulation of the horseshoe crab fishery, which resulted in a horseshoe crab harvesting ban for all but biomedical use.

South Carolina Code of Law, Section 50-5-1330

1994

LAL methodologies advanced from gel clot and turbidimetric to the colorimetric techniques of endpoint and kinetic chromogenic in the late 1980s and early 1990s. 

Berzofsky, R.N. (1994)  Kinetic Assay for Endotoxin Using Limulus Amebocyte Lysate and Chromogenic Substrate, United States Patent, 5,310,657

1998

The Atlantic States Marine Fisheries Commission (ASMFC) establishes a formal fisheries management plan for horseshoe crab populations’ coast wide.

 

2000

Jay Harrington (horseshoe crab fisherman) sues the US Department of Interior on behalf of Associates of Cape Cod, Inc. to continue to allow the harvest by special permit of horseshoe crabs in the Monomoy NWR (Cape Cod). An injunction is issued to his benefit allowing continued harvest for two seasons but final ruling closes harvest of horseshoe crabs in the NWR permanently. Although harvest pressure is moved to other areas of Cape Cod and Southeastern MA, horseshoe crab numbers today (2013) are higher than any other area in New England.

 

2000

The Cape Cod (MA) National Seashore closes waters in its boundaries to the harvest of horseshoe crabs. Although other fishing activities in the National Park are allowed by charter, e.g. shellfishing, the Park designates the horseshoe crab as “wildlife” and not a “shellfish” thus extending its protection (even for biomedical use and with conditions that bled crabs be returned to the area) in the Park. The largest area affected is a part of Pleasant Bay, MA, another Harrington fishing location.

 

2001

Carl N. Shuster Sanctuary established (no harvesting of horseshoe crabs for any reason within the sanctuary boundaries).

 

2001

There are four (4) companies licensed by the FDA in the United States to manufacture and market LAL commercially. These were (with their accompanying areas of horseshoe crab harvest):

  • Associates of Cape Cod, Inc., Woods Hole, MA (Cape Cod, Southeastern Massachusetts, Rhode Island)

  • Mallinckrodt Inc., St. Louis, MO (Various locations in North Carolina)

  • BioWhittaker (originally Microbiological Associates, acquired by Cambrex in 1997) (Chincoteague, VA)

  • Haemachem, St. Louis, MO (Various locations in North Carolina and Long Island, NY)

  • Charles River Endosafe (originally Endosafe, Inc. acquired by Charles River Laboratories in 1995) (Various locations in South Carolina and contract processed blood from Cape May, New Jersey via Limuli Laboratories, Inc., an outgrowth of Marine Biologicals Inc., at one time licensed by the FDA to produce a final LAL product.)

Baxter Travenol, Deer Lake, IL, still retains a license to manufacture LAL and use the product in their own facility but do not market LAL commercially.

 

2001

An alternative method for endotoxin detection that used a recombinant form of Factor C (rFC) from the horseshoe crab was introduced by the National University of Singapore.

Ding, JL and B. Ho (2001) A New Era in Pyrogen Testing, Trends in Biotechnology, Vol 19, No 8. 

2003

Cambrex BioScience (formerly BioWhittaker) licenses the rFC technology from the National University of Singapore, and commercializes their rFC product PyroGene™.

 

2006

The Endosafe®-PTS™ for endotoxin testing is licensed by the FDA after three years of use in R&D application. Conforming to the FDA initiative for process analytical technology (PAT), the portable incubating spectrophotometer provides rapid endotoxin testing results at the point of sample collection, contributing to improved quality, safety and efficiency in the manufacturing process.

Charles River launched the Endosafe®-PTS™ aboard the International Space Station, where astronauts began using the system to perform biological studies necessary for an extended human presence in space–from crew health and spacecraft environmental studies to the search for life elsewhere in the solar system. NASA used the Endosafe®-PTS™ to monitor the environment for microbial contamination during the construction of the Mars exploration rovers "Spirit" and "Opportunity". Future applications of PTS may include monitoring the spread of Earth-derived biological material to the Moon and detecting signs of microbial life on Mars.

 

2011

In July, FDA withdraws the 1987 LAL Guidance document, stating that it was obsolete and would be replaced in the future.

 

EndoLISA, innovative ELISA-test using an Endotoxin-specific phage protein for binding and rFC for detecting endotoxin is introduced by Hyglos GmbH of Munich, Germany

 

2012

In June, FDA issues the less-prescriptive Guidance for Industry - Pyrogen and Endotoxins Testing: Questions and Answers as a replacement to the 1987 Guidance document.  The new document states the use of recombinant Factor C methods is accepted by FDA if validated as per USP General Chapter <1225> Validation of Compendial Methods.

 

2013

In January, Hyglos launches their recombinant Factor C (rFC) endotoxin detection assay EndoZyme®.

 

2013

There are still four companies licensed by the FDA to produce LAL in the US:

  • Associates of Cape Cod, Inc., Falmouth, MA now wholly owned by the Tokyo-based Seikagaku Corporation

  • Charles River Endosafe, Charleston, SC

  • Lonza, a Swiss-based company with LAL facilities in Walkersville, MD (formerly BioWhittaker/Cambrex, also includes original technology from Mallinckrodt, Inc. no longer in the business themselves)

  • WAKO Pure Chemical Company, an Osaka-based company that acquired Haemachem, Inc. with a US base in Richmond, VA and an LAL facility on the VA shore.

 

2014

In August, Hyglos introduces the Endo-RS, Endotoxin Recovery Kit, a sample preparation method for biopharmaceutical formulations affected by the Low Endotoxin Recovery (LER) phenomenon.

 

2016

In July, the European Pharmacopoeia publishes the revised Chapter 5.1.10, Supplement 8.8, including Recombinant Factor C (rFC) as an alternative method since, “this practice avoids the use of animal species“.

"The use of alternative reagents such as recombinant factor C as a replacement to the amoebocyte lysate eliminates the use of a reagent extracted from live animals. Replacement of a rabbit pyrogen test or a bacterial endotoxin test prescribed in a monograph by a test using recombinant factor C reagent or any other reagent as a replacement of the amoebocyte lysate is to be regarded as the use of an alternative method in the replacement of a pharmacopoeial test, as described in the General Notices.”

 

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