Revolution in Bacterial Endotoxin Test (BET)

Background:

Definition:

The Bacterial Endotoxins Test (BET) is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).

Revolution in BET test from discovery to yet:

A. 1885

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

Observations upon the chemical composition and coagulation of the blood of Limulus Polyphemus and Callinectes hastatus.

B. 1953

Frederik B. Bang describes the effects of injecting a marine bacterium into Limulus polyphemus. His results indicate this causes intravascular clotting and other Gram-negative bacteria could cause similar results but Gram-positive bacteria did not produce this effect. 

This finding was the foundation which ultimately lead to the discovery of LAL many years later.

C. 1956

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

D. 1964

Discovery that endotoxin is the key factor in clotting of Limulus blood and the role of endotoxin in the extracellular coagulation of Limulus blood. 

E. 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.

F. 1972

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

G. 1973

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

Food and Drug Administration proposes standards for the manufacture of LAL. 

H. 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.

I. 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.

J. 1978

FDA proposal for the live release of horseshoe crabs back to their native environment after only one blood collection. 

K. 1980

The United States Pharmacopeial Convention (USP) publishes General Chapter <85> Bacterial Endotoxins Test in pharmaceutical products and medical devices.

L. 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.

M. 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. 

N. 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.

O. 2011

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

P. 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.

Q. 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.”

R. 2018

FDA approves first drug using the recombinant Factor C (rFC) Assay for endotoxin testing of Eli Lilly’s Emgality™ , the first drug approved by the U.S. Food and Drug Administration (FDA) to have been released using this method instead of traditional Limulus Amebocyte Lysate (LAL) based methods.

There are three techniques for this test as discuss above: 

1. Gel clot technique:

It is based on clotting of the lysate reagent in the presence of endotoxins.

The minimum concentration of endotoxin required to cause the lysate to clot under standard conditions is the labeled sensitivity of the lysate reagent.

2. Turbidimetric technique:

It is based on the development of turbidity after cleavage of an endogenous substrate.

Depending on the test principle used, this technique is further classified as either endpoint- turbidimetric or kinetic-turbidimetric. 

A.Endpoint-turbidimetric technique:

It is based on the quantitative relationship between the concentration of endotoxins and the turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period. 

B. Kinetic-turbidimetric technique:

It is a method to measure either the onset time needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development.

3. Chromogenic technique:

It is based on the development of color after cleavage of a synthetic peptide-chromogen complex.

The chromogenic method measures the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the LAL Reagent. 

Depending on the test principle employed, this technique is further classified as either endpoint-chromogenic or kinetic-chromogenic. 

A. Endpoint-chromogenic technique: 

It is based on the quantitative relationship between the concentration of endotoxins and the release of chromophore at the end of an incubation period. 

B. Kinetic- chromogenic technique:

It is a method to measure either the onset time needed to reach a predetermined absorbance of the reaction mixture or the rate of color development.

Alternative Endotoxin Testing Methods:

There are alternative and sustainable methods available for those pharmaceutical, medical device, and dialysis companies willing to embrace sustainable, animal-friendly innovations in endotoxin detection.

1. Recombinant Factor C (rFC) Methods derived from the Horseshoe Crab:

The National University of Singapore developed and Lonza commercialized an endotoxin test that does not require horseshoe crab blood. Instead, the DNA for one of the horseshoe crab blood clotting factors, Factor C, was cloned and is manufactured recombinantly (synthetically). Recombinant Factor C (rFC), similar to the native Factor C, is activated by endotoxin.

In the rFC test method, branded PyroGene™ by Lonza, activated rFC cleaves a fluorogenic substrate and the fluorescent signal is monitored and analyzed to quantitate endotoxin content.

The horseshoe crab provides some DNA to support this alternative advancement in endotoxin detection.

In world, horseshoe crabs are limited and/or declining, switching to the rFC method could help make a positive impact to the crab population.

The methods based on rFC have included in the European Pharmacopoeia guidelines in July 2020 as valid alternatives to the LAL test. In order to be used for product release, the rFC methods must be validated and show to provide equivalent or better performance than LAL.

2. Monocyte Activation Test:

Another method that does not require the use of horseshoe crab blood is the Monocyte Activation Test (MAT) or the In Vitro Pyrogen test.  

This test method uses human blood rather than horseshoe crab blood.  The MAT method measures the release of cytokines from blood cells due to the presence of pyrogens, such as endotoxin, in the test sample.  

The MAT is basically mimicking what occurs in our blood stream when it is exposed to pyrogenic substances.  

The MAT has an advantage over the LAL/TAL and rFC methods as it can detect non-endotoxin pyrogens. 

Monocyte activation tests (MATs) are human cell-based tests to detect and quantify pyrogens. MATs use an ELISA assay to measure cytokine release from treated blood cells.

Testing time duration of different test:

1. Rabbit Pyrogen Test:

 It is performed in 3 hours (after pre-test preparation)

2. LAL and rFC Test:

It is performed in about 2 hours

3. Monocyte Activation Test (MAT):

It is performed in 24 hours

Is Validation a factor in choosing what test to perform?

1. Rabbit pyrogen test – nominal

2. Bacterial endotoxin test - product-specific, regulatory approval

3. rFactor C test - product-specific, regulatory approval

4. Monocyte Activation Test - product-specific, regulatory approval

Reference:

1.https://www.horseshoecrab.org/med/timeline.html

2. USP <85> Bacterial Endotoxins Test in pharmaceutical products and medical devices.

3. USP <151> Pyrogen Test.

About the Author:

Dhansukh Viradiya is a highly accomplished expert in the pharmaceutical and biopharmaceutical industries. With over 10 years of experience in the field, he has gained comprehensive knowledge and expertise in various areas, including Process Validation, Cleaning Validation, Quality Management System, In-process quality assurance, Qualification etc.

Mr. Dhansukh holds a Master's degree in Pharmacy from a renowned University, where he specialized in Quality Assurance. 

As a thought leader, Mr. Dhansukh has published numerous articles and white papers on various topics related to pharmaceutical and biopharmaceutical industries. His research work focuses on emerging trends, current regulatory expectations, advancements in technology, personalized medicine, and the intersection of healthcare and technology.

With his passion for improving patient care and dedication to advancing the field, Dhansukh Viradiya continues to make significant contributions to the pharmaceutical and biopharmaceutical industries. His insights and expertise make him a valuable resource in understanding the dynamic landscape of these sectors and their impact on global healthcare.

Disclaimer:

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