Endotoxin Testing Service
  • OVERVIEW

Endotoxin Testing


Endotoxins are large and complex toxic molecules of polysaccharide, lipid A, and other cell wall components of Gram-negative bacteria. Endotoxin testing of biologics prepared either in bacteria or in mammalian cells is a necessity to ensure that the therapeutic product is endotoxin free. Seattle Genova provides endotoxin testing for pharmaceutical, biopharmaceutical, medical device, research, and other industries. We are offering endotoxin test with Limulous amoebocyte lysate (LAL), Recombinant Factor C (rFC) method, Chrmogenic LAL Endotoxin Test, Turbidimeteric LAL Endotoxin Test, Rabbit Pyrogen Test and Monocyte Activation Testing.


Gel-Clot Method (LAL Test)

This is a qualitative method based on a gel clot formation when endotoxin interacts with Limulus amebocyte lysate (LAL). It is based in the biology of the horseshoe crab which produces LAL enzymes in blood cells to bind and inactivate endotoxin from invading bacteria which forms a gel-like clot when incubated in the presence of endotoxins. This method is used to determine if products or materials are "endotoxin free". The sensitivity or detection limit of the endotoxin test is 0.06 EU/ml (Endotoxin Units).


Recombinant Factor C (Rfc) Method

The rFC method is used for detecting endotoxin in environmental and biological samples and medical devices. Advantages of the fluorescence-based rFC assay include: Endotoxin specificity - eliminate the potential for false positives due to Beta-(1,3)-glucans. Assay components are derived from recombinant source, offering a sustainable and reliable method. Better lot-to-lot consistency of assay components. Increased precision and accuracy. FDA approved as a final release test.

Chrmogenic LAL Endotoxin Test Method

This method is based on the development of color after cleavage of a synthetic peptide-chromogen complex. The kinetic chromogenic endotoxin test is a reaction between bacterial endotoxin present in a test material and synthetic chromogenic LAL reagents. Color changes if endotoxin is present in the test material. Test sensitivity depends upon the specific lysate used. Lowest detection limit in case of kinetic chromogenic test is 0.005 EU/ml.

Turbidimeteric LAL Endotoxin Test Method

This method is based on the development of turbidity after cleavage of an endogenous substrate. It determines the cloudiness of the solution. If endotoxin is present, clotting of the solution results in cloudiness which reduces the transmittance of light when observed under spectrophotometer. Test sensitivity depends upon the specific lysate used. Lowest detection limit in case of turbidimeteric endotoxin test is 0.005 EU/ml as compared to 0.03 EU/ml in case of gel clot endotoxin test method.

Rabbit Pyrogen Test

The Rabbit Pyrogen Test is an in vivo test to detect pyrogens qualitatively. Rabbits have a similar pyrogen tolerance to humans, so by observing a change in body temperature in rabbits it is possible to decide of the presence of pyrogens. This method can detect non-bacterial endotoxin pyrogens as well as bacterial endotoxins.


Monocyte Activation Testing

Monocytes, the key cells of innate immunity, respond to the presence of pyrogens (endotoxins and non-endotoxin pyrogens) by secreting pro-inflammatory cytokines, which can be measured by an ELISA assay. The overall procedures are: 1. Your medicinal product sample is incubated with peripheral blood mononuclear cells (PBMC). 2. Overnight, the toll-like receptors (TLR) of the monocytes recognise the presence of pyrogens and activate the signaling pathways that launch the human immune system response. In turn, this stimulates cytokine release. 3. The next morning, a human IL-6 ELISA is used as a readout to detect and quantify the concentration of released cytokines. 3. Using the LPS standard curve, the quantified cytokine concentration is finally converted to the Endotoxin Equivalent Units/ml. If below the product's CLC, the MAT is passed. The MAT assay is used to predict the potential of a product to elicit a fever response in human recipients.

Sample Requirements

USP requires 3% of the production lot with a minimum of 3 and maximum of 10 devices to be pooled and tested. For liquid samples, a minimum of 5 ml is required and powder samples require enough material to reconstitute into a minimum of 5 ml pyrogen-free water. Initial validation on 3 lots must be performed in order to validate the method of testing for a given product.

Endotoxin Testing


Endotoxins are large and complex toxic molecules of polysaccharide, lipid A, and other cell wall components of Gram-negative bacteria. Endotoxin testing of biologics prepared either in bacteria or in mammalian cells is a necessity to ensure that the therapeutic product is endotoxin free. Seattle Genova provides endotoxin testing for pharmaceutical, biopharmaceutical, medical device, research, and other industries. We are offering endotoxin test with Limulous amoebocyte lysate (LAL), Recombinant Factor C (rFC) method, Chrmogenic LAL Endotoxin Test, Turbidimeteric LAL Endotoxin Test, Rabbit Pyrogen Test and Monocyte Activation Testing.


Gel-Clot Method (LAL Test)

This is a qualitative method based on a gel clot formation when endotoxin interacts with Limulus amebocyte lysate (LAL). It is based in the biology of the horseshoe crab which produces LAL enzymes in blood cells to bind and inactivate endotoxin from invading bacteria which forms a gel-like clot when incubated in the presence of endotoxins. This method is used to determine if products or materials are "endotoxin free". The sensitivity or detection limit of the endotoxin test is 0.06 EU/ml (Endotoxin Units).


Recombinant Factor C (Rfc) Method

The rFC method is used for detecting endotoxin in environmental and biological samples and medical devices. Advantages of the fluorescence-based rFC assay include: Endotoxin specificity - eliminate the potential for false positives due to Beta-(1,3)-glucans. Assay components are derived from recombinant source, offering a sustainable and reliable method. Better lot-to-lot consistency of assay components. Increased precision and accuracy. FDA approved as a final release test.

Chrmogenic LAL Endotoxin Test Method

This method is based on the development of color after cleavage of a synthetic peptide-chromogen complex. The kinetic chromogenic endotoxin test is a reaction between bacterial endotoxin present in a test material and synthetic chromogenic LAL reagents. Color changes if endotoxin is present in the test material. Test sensitivity depends upon the specific lysate used. Lowest detection limit in case of kinetic chromogenic test is 0.005 EU/ml.

Turbidimeteric LAL Endotoxin Test Method

This method is based on the development of turbidity after cleavage of an endogenous substrate. It determines the cloudiness of the solution. If endotoxin is present, clotting of the solution results in cloudiness which reduces the transmittance of light when observed under spectrophotometer. Test sensitivity depends upon the specific lysate used. Lowest detection limit in case of turbidimeteric endotoxin test is 0.005 EU/ml as compared to 0.03 EU/ml in case of gel clot endotoxin test method.

Rabbit Pyrogen Test

The Rabbit Pyrogen Test is an in vivo test to detect pyrogens qualitatively. Rabbits have a similar pyrogen tolerance to humans, so by observing a change in body temperature in rabbits it is possible to decide of the presence of pyrogens. This method can detect non-bacterial endotoxin pyrogens as well as bacterial endotoxins.


Monocyte Activation Testing

Monocytes, the key cells of innate immunity, respond to the presence of pyrogens (endotoxins and non-endotoxin pyrogens) by secreting pro-inflammatory cytokines, which can be measured by an ELISA assay. The overall procedures are: 1. Your medicinal product sample is incubated with peripheral blood mononuclear cells (PBMC). 2. Overnight, the toll-like receptors (TLR) of the monocytes recognise the presence of pyrogens and activate the signaling pathways that launch the human immune system response. In turn, this stimulates cytokine release. 3. The next morning, a human IL-6 ELISA is used as a readout to detect and quantify the concentration of released cytokines. 3. Using the LPS standard curve, the quantified cytokine concentration is finally converted to the Endotoxin Equivalent Units/ml. If below the product's CLC, the MAT is passed. The MAT assay is used to predict the potential of a product to elicit a fever response in human recipients.

Sample Requirements

USP requires 3% of the production lot with a minimum of 3 and maximum of 10 devices to be pooled and tested. For liquid samples, a minimum of 5 ml is required and powder samples require enough material to reconstitute into a minimum of 5 ml pyrogen-free water. Initial validation on 3 lots must be performed in order to validate the method of testing for a given product.

Request Quote
First Name *
Last Name *
Email Address *
Instiution *
Prodcuts/Services *
Questions & Comments *