BET Products

LAL Reagents

Introduction to the LAL Test

Limulus Amebocyte Lysate (LAL) tests detect and quantify bacterial endotoxins extracted from the outer membrane of gram negative bacteria. The critical component of the LAL reagents used in endotoxin tests is derived from blood cells (amebocytes) of the horseshoe crab, Limulus polyphemus. It contains the proteins of the blood clotting mechanism, which is triggered by endotoxins. LAL reagents are primarily used to test for endotoxins in injectable pharmaceuticals, biological products, and medical devices. They are also used in renal dialysis centers and a wide range of other applications. LAL tests are described in the Bacterial Endotoxins Test chapter in the United States Pharmacopeia (Chapter <85>) and in the equivalent chapters in the European Pharmacopoeia (Chapter 2.6.14) and the Japanese Pharmacopoeia (General Tests, No. 4.01).

LAL Test Methods

There are three principal LAL test methods: the gel-clot, turbidimetric and chromogenic methods. The latter two may be grouped together as photometric methods as they require an optical reader.

Chromogenic Method

The LAL reagent is formulated with a synthetic substrate which produces a chromophore when cleaved by endotoxin activated enzyme. The test is read at 405 nm, usually in a microplate reader. The maximum sensitivity of 0.001 EU/mL can be achieved when using ACC's Pyrochrome® Chromogenic reagent with Glucashield® Buffer.

• Requires a tube reader or incubating microplate reader (an incubating reader is required for the kinetic method)
• Maximum sensitivity to 0.001 EU/mL when using ACC's Pyrochrome® reagent with Glucashield® Buffer
• A quantitative assay providing electronic stored data and print-outs of results
• Incubation time varies depending on the standard curve range
• Higher sensitivity allows for greater dilution to overcome interference
• The option of a diazo-coupled endpoint method (read at 540–550 nm) is available, which is useful for samples that absorb light at 405 nm

Turbidimetric Method

The optical density (turbidity) increase that accompanies the clotting reaction is read in the Pyros Kinetix® Flex tube reader or in a incubating microplate reader. The maximum sensitivity of 0.001 EU/mL can be achieved when uising ACC's Pyrotell®-T reagent in our Pyros Kinetix® or Pyros Kinetix® Flex tube reader. 

• Requires the Pyros Kinetix® Flex tube reader system or an incubating microplate reader
• Maximum sensitivity to 0.001 EU/mL, highest level of sensitivity available in the LAL industry
• Incubation time varies depending on the standard curve range
• High sensitivity allows for greater dilution to overcome interference

Gel-clot Method

This is the simplest LAL test. The formation of a gel-clot indicates the presence of endotoxin in a sample. The method is performed in small test tubes and is read manually by inverting the test tubes. The maximum sensitivity is 0.03 EU/mL.

• Requires non-circulating water bath or dry bath incubator
• Manually read test
• Reagents of differing sensitivity available: 0.5 (STV only), 0.25, 0.125, 0.06 and 0.03 EU/mL
• May be less sensitive to interference than other LAL methods
• Is the "gold standard" for the great majority of products in the United States, European and Japanese pharmacopeia (i.e. it is the default method used to resolve a dispute)

Selecting an LAL method?

When deciding which LAL test method is to be used, the following questions can be asked:

• What is the available budget?
• What are the regulatory requirements, if any?
• What type of product or sample is going to be tested?
• What test sensitivity is required? (What is the endotoxin limit specification for the sample?)
• Is electronic storage of data desired?

If the budget is limited, the gel-clot method is the least expensive method as no optical reader is required. Also, the gel-clot method may be the method of choice for opaque samples, suspensions or colored samples, though dilution of the sample may enable use of the photometric methods. For users with a non-incubating optical reader, an endpoint chromogenic test may be the best choice. As with other photometric methods, this will provide printouts of results and electronic storage of data. The kinetic methods (chromogenic and turbidimetric) provide the widest detection range and sophisticated software. The photometric methods offer the greatest sensitivity, allowing detection of low endotoxin concentrations and greater dilutions of sample, which is important for overcoming interference.

Overview of Testing Procedures

The following section summarizes the procedures/steps to be taken to perform routine product release testing of a sample in a regulated environment. In an unregulated environment, or when testing for informational purposes only, follow the procedures described under Product Characterization.

Qualification of Reagent, Technician and Laboratory

The reagent must be tested to ensure that it is performing to specification, technicians must be qualified to perform the test and the absence of significant day to day or inter-technician variability in the laboratory should be documented. This requires tests using endotoxin standards only, not samples.

Product Characterization

Samples should be characterized for endotoxin contamination and/or potential interference. Characterization is not a regulatory requirement, but is important to develop a test method that can be validated to demonstrate the absence of interference. It is typically performed by testing a series of dilutions of sample without and with a known amount of added endotoxin (Positive Product Control or PPC). The purpose of the PPC is to indicate that added endotoxin is appropriately detected and that the sample does not interfere with the test. From the results of characterization testing, a product dilution (and possibly product treatment) is selected for validation of the test (see below). The endotoxin limit for the product must be detectable at the dilution selected.

Test for Interfering Factors (Validation)

Inhibition/Enhancement testing is performed to validate the test for the particular sample type. It is accomplished by demonstrating, with three lots of product, that endotoxin added to the sample in PPCs can be readily detected within required limits.

Routine Testing

Routine testing is conducted at the validated dilution and includes a parallel PPC to control for interference. Tests should also include negative controls and appropriate standards. A minimum of three units per lot of drug product should be tested, with the samples taken from the beginning, the middle and the end of the production run. For medical devices, aqueous extracts of up to ten units are tested, usually after pooling.

Conclusion

The preceding overview is just the beginning. Further assistance with selecting a test method or reagent sensitivity is always available from our technical support personnel, and representatives in the field. The LAL Update, our newsletter, includes useful technical articles and is available on the website. Our Contract Test Service regularly assists with characterization and method development and can provide results by all test methods. Whichever method is selected, you can always be assured of the full support of Associates of Cape Cod, Inc.

For details on the regulatory requirements for compliant LAL testing in the United States (US), users should consult the current revision of the United States Pharmacopeia (USP), chapter <85>, "Bacterial Endotoxins Test."