Endotoxin Testing

overview

Endotoxins are of great concern in the medical device and pharmaceutical industries and, due to the implications of their presence, are  currently  a growing concern in the biotech and research fields. An endotoxin is  a pyrogen, which is defined as a substance that can cause a fever response. They are also toxic to cells grown in tissue culture conditions.

Endotoxins are found in the lipopolysaccharide complex of the outer membrane of Gram negative bacteria. When the cell dies, the membrane of the cell ruptures and endotoxins are released into the surrounding environment. This can cause contamination that may have adverse effects on a variety of applications and experiments.

 

 

Why certify?

Endotoxins are found in the air, water, soil, via human contact, and any non-sterile environment.

Endotoxin contamination can affect general laboratory consumables. Certifying products endotoxin free has become a common practice among general laboratory plastics manufacturers as well as medical and drug manufacturers. This is due to the ability of endotoxins to interfere with many common laboratory techniques such as: the creation and maintenance of cell culture lines, and the transfection of cell lines with molecular reagents. If the product is not certified to be free of endotoxins, many experiments may fail.

The presence of endotoxins is an indication of the overall purity and microbial activity of a particular product or raw material. Testing your product lines for this common contaminant adds integrity to your products and allows a wider range of customers to utilize your products for their experiments and applications.

 

 

Endotoxin/pyrogen
Free certification assay

ARYTE Bioscience and all its test operators have been qualified according to USP guidelines to utilize the LAL (Limulus Amebocyte Lysate) Gel Clot method to test raw materials or end products for the presence of endotoxins. This method uses components found in the blood of the Blue Horseshoe Crab, Limulus polyphemus, which forms a gel-like clot when incubated in the presence of endotoxins. This method is used to:   

  • “Spot Test” a raw material or product and quantify the endotoxin levels.(LAL Preliminary Test)

  • perform Non-Pyrogenic Validations as required by the USP in order to advertise a product as being Pyrogen Free or Non-Pyrogenic.(LAL Validation Assay)

  • perform Routine lot tests on product batches to determine if products or materials are “Endotoxin Free”.           (Routine LAL Assay)

The sensitivity or detection limit of the Gel Clot endotoxin test is 0.06 EU/ml (Endotoxin Units). Products and materials can be certified to a sensitivity of 0.03 EU/ml upon request

0.005 -0.001 EU/mL sensitive tests can be run with advanced notice.

test methods

Upon receiving the product to be tested ARYTE one of the three Test Methods is employed.


 

LAL Preliminary assay

When using the LAL Gel Clot method, the quantitative LAL assay is usually used on liquid products in which a Preliminary Assay and a Validation of a dilution series is performed. Although not common, the quantitative assay can be performed on devices. The Preliminary Assay tests for the pH of the product. The pH must be compatible with the LAL gel clot assay in order for a validation to be performed. This Validation tests the product dilution series for an endpoint which is used to calculate the final range of endotoxin concentration present in the product.

 

LAL Validation assay

Non-Pyrogenic Validation is a requirement of the FDA in order to advertise a product as being Pyrogen Free or Non-Pyrogenic. This test consists of testing in quadruplicate three separate lots of the same product.  Product extracts are made using the three separate lots. The product extracts are used to prepare three sets of standard series of the Control Standard Endotoxin (CSE). Each dilution of the standard series is run in quadruplicate. The product extract series are incubated along with a standard series of the CSE. After the incubation period, the tubes containing the controls and the extract are observed for the presence of the gel clot.

 

If the unspiked product samples are all negative and the product series Endotoxin concentration is between 0.03 Eu/ml and 0.12 Eu/ml for all three lots then the product is not introducing any inhibitory or enhancing effects. The LAL validation is a one time procedure and does not need to be repeated unless there are changes in manufacturing procedures, raw materials or product formulations.

 

Once it is determined that the product does not have any inhibitory or enhancing effects the product is validated. After product validation, each lot that will be advertised as Non-Pyrogenic must be routinely tested in accordance with FDA guidelines.

 

Routine LAL
assay

Routine LAL testing involves the testing of each lot of product following an LAL Validation. For this test, a  product extract is made, then the product is exposed to and incubated with the horseshoe crab lysate (LAL). The product extract is incubated along with a standard series of the Control Standard Endotoxin (CSE) as the positive control and the unexposed extract fluid as a negative control. After the incubation period, the tubes containing the controls and the extract are observed for the presence of the gel clot. A clot indicates the presence of endotoxins. No clot indicates absence of endotoxins (i.e. < 0.06 EU/ml).

The Routine LAL assay is a qualitative test utilizing the LAL Gel Clot method to test the endotoxin levels of the product to be either greater than or less than the sensitivity of the test, which is 0.06 EU/ml. This type of test can be performed on solid and liquid products.

Once the certification assay is completed, the customer is contacted with the preliminary results if indicated and a report containing a Data and Results sheet and a Certificate of Analysis is mailed to the customer.

Non-Pyrogenic certification testing consists of device extraction or liquid, controls and inhibition/enhancement using the LAL gel clot method. Additional fees apply for quantitative testing.