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Bacterial Endotoxins Testing

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USP 85 – Bacterial Endotoxin Test (BET)

Introduction to USP 85 – Bacterial Endotoxin Test (BET)

USP 85 Bacterial Endotoxins Test (BET) which is commonly known as the LAL (Limulus Amebocyte Lysate) test, is an important assay for detecting endotoxins to ensure the safety of pharma products and medical equipment. 

Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria. They are released when bacteria disintegrate or during bacterial growth. Sterilization processes that usually kill bacteria might not be effective enough to deactivate endotoxins. Therefore, products must be effectively tested to ensure that they are free from any remnants of endotoxins, which might pose a risk.

Compliance and Industry Standards for USP 85 Bacterial Endotoxin Testing

Pharmaceutical manufacturers and developers of medical devices have to comply with numerous stringent standards such as USP 85, to meet high quality and safety standards. Compliance with this standard ensures that tested products do not contain endotoxins, thereby protecting patients from health risks. 

Testing also helps meet the standards set by health authorities like the U.S. Food and Drug Administration (FDA), the European Pharmacopeia (EP), and other global regulatory bodies. Not adhering to this standard can lead to extreme penalties, including product recall, imposition of fines, and serious health risks to patients.

Bacterial Endotoxin Testing is required for a wide scope of products, which includes:

  • Parenteral drugs
  • Biological products
  • Vaccines
  • Implantable medical devices
  • Dialysis solutions 

Guidelines for Conducting USP 85 Bacterial Endotoxin Testing 

  • The sensitivity of the lysate needs to be confirmed with every batch used. The labeled sensitivity, λ, has to be within the valid range (0.5λ – 2λ) usually verified by conducting tests using standard endotoxin solutions.
  • A preparatory test is required to ensure that the sample does not contain any substances that will interfere with the detection of endotoxin. 
  • The test should be carried out at the incubation temperatures specified by the manufacturer. In the case of the gel clot method, incubation is usually done at 37 ± 1°C for 60 ± 2 minutes.
  • The test’s lysate sensitivity and accuracy must be verified using standard endotoxin solutions as positive controls.
  • If interference is detected, then it should be eliminated from the sample using methods such as dilution, filtration, neutralization, or heating.

Test methodology

Endotoxin testing methods entail the use of Limulus Amebocyte Lysate (LAL), which is extracted from the blood cells of the horseshoe crab, Limulus polyphemus. This lysate is endotoxin-sensitive and forms the basis of the test techniques detailed below.

Gel-clot technique

This technique is based on the clotting of the lysate reagent in the presence of endotoxins. In this assay, a clot forms in the bottom of the test tube in the presence of endotoxins above a specified threshold. The minimum endotoxin level required to form the clot is termed as the labeled sensitivity of the lysate. This test is qualitative, meaning it only confirms the presence of endotoxins but does not provide its concentration. 

Photometric techniques

Photometric methods are quantitative and include two main types:

  • Turbidimetric: This technique measures the increases in turbidity of the reaction mixture caused by endotoxins. There are two types:
  • Endpoint-turbidimetric assay: The endpoint of the reaction is measured; a quantitative result is obtained.
  • Kinetic-turbidimetric assay: The rate or time at which a certain level of turbidity is obtained is measured.
  • Chromogenic:The Chromogenic technique measures the color change that occurs when endotoxins react with a reagent added to LAL. It may also be carried out in two variants:
  • Endpoint-chromogenic assay: The color produced is measured at the end of incubation.
  • Kinetic-chromogenic assay:  The rate of color development is monitored.

Applications and Importance of USP 85 Endotoxin Testing

Bacterial endotoxin testing is an essential component in monitoring the quality as well as safety of manufactured products such as pharmaceuticals and medical devices.  Here’s a detailed look at the various applications and why this testing is indispensable.

Injectable pharmaceuticals

In the case of injectable drugs, whether vaccines or biologics, endotoxins can cause severe immunologic reactions. The BET test ensures that these products are not contaminated with endotoxins that cause side effects such as fever, inflammation or even septic shock in individuals.

Medical devices

Medical devices that have direct or indirect contact with the cardiovascular system, central nervous system, or those devices that are implantable must undergo very strict endotoxin testing. This entails products like catheters, stents, and implants. BET makes certain that these devices will not trigger an immune response that will result in the development of complications or device rejection.

Intravenous solutions

The fluids to be administered intravenously have to be endotoxin-free to prevent pyrogenic reactions in patients.

Surgical and procedural kits

Products used in performing medical procedures or surgeries may have parts which come into contact with body tissues. These materials must be carefully checked for endotoxins since postoperative infections and complications due to endotoxins are extremely dangerous.

Ophthalmic products

Products used in ophthalmology such as eye drops and contact lens solutions, require the endotoxin level to be very low since the eye is sensitive and prone to infection. Endotoxin testing of these products is useful in reducing incidences of eye injuries including irritation and other severe eye complications.

Transdermal patches

Transdermal drug delivery systems deliver medication through the skin. As these patches are an entryway to blood, it becomes necessary to screen for endotoxins in order to prevent skin reactions and systemic toxicity.

Dialysis equipment

In the case of patients on dialysis, it is important that all equipment employed is endotoxin free particularly the dialysate and other fluids. Endotoxins may cause adverse effects including fever and hypotension and these are potentially fatal in renal patients.

Biotechnology products

Biotechnological products, especially those produced using bacterial cultures, may be prone to endotoxin contamination. Endotoxin testing is vital to ensure that these products, which include enzymes and recombinant proteins, are safe for human use.

Advantages of Endotoxin Testing

Endotoxin testing offers several advantages:

  • Sensitivity

The test detects the lowest levels of endotoxins, which is important for product safety.

  • Versatility

The test can be applied to a diverse range of product types, including liquid, solid, and devices.

  • Acceptance by regulatory bodies

Results obtained by performing this test are generally accepted by most regulatory bodies worldwide. Hence, the product is acceptable in international markets.

Frequency and Timing of USP 85 Bacterial Endotoxin Testing 

The frequency of testing for bacterial endotoxin varies from product to product and its intended application. In general, testing must be performed:

  • In the development phase to ensure that raw materials and intermediates must be free from endotoxin contamination.
  • As a final quality control check to ensure that the finished product conforms to regulatory limits for endotoxins.
  • Periodic testing for the presence of endotoxins in products under ongoing production to ensure that contamination does not occur during production processes. 

Conclusion: Ensuring Safety Through Endotoxin Testing 

Endotoxin testing is the core of quality assurance for pharmaceutical products, biologics, and medical devices. It is designed to ensure that the product is safe for patients through rigorous testing for the presence of endotoxins. As endotoxins are known to cause serious reactions, it is a regulatory requirement and a moral obligation to test products under this standard in order to protect patient health.

Being one of the renowned testing labs for USP 85, we offer a range of testing services to guarantee optimal safety of drugs and medical devices. Our laboratories are well-equipped to ensure efficiency, accuracy and timely production of test results.

Our state-of-the-art laboratories are equipped with advanced technology to ensure accurate and reproducible results within the designated time frame.

If you are looking to ensure your products meet the rigorous standards for endotoxin testing, contact MIS today to learn more about our Bacterial Endotoxin Testing services and how we can assist you in maintaining the highest quality and safety standards in your product offerings.

FAQs

USP 85 provides methods for the detection and quantification of endotoxin contamination in pharmaceutical products, medical devices, and similar materials. In USP 85, product samples are screened for endotoxins by using Limulus Amebocyte Lysate (LAL) test. LAL test can be performed using three techniques: Gel-clot, Turbidimetric, and Chromogenic Standard Endotoxin Stock Solution.

USP 85 test is applicable to test medical devices, intravenous solutions, opthalmic products, injectable medications and other biological products.

It takes 3-4 weeks to complete the USP 85 test.

Endotoxin testing is a laboratory method employed in the identification and quantification of endotoxins that are derived from the cell walls of gram-negative bacteria, specifically lipopolysaccharides. The main objective of endotoxin testing is to ensure that the product does not contain any toxic levels of endotoxin that will harm the patient.

The Bacterial Endotoxin Test (BET) or Limulus Amebocyte Lysate (LAL) test is a routine assay that measures the endotoxins. The test employs the principle that the blood of horseshoe crabs coagulates when endotoxins are present. There are three main types of LAL tests: gel-clot, turbidimetric and chromogenic methods.

Endotoxin testing plays a vital role in eliminating the risks of use of contaminated pharmaceuticals, medical devices, and biologics. Endotoxins can act as pyrogens, which are agents that can induce fever, shock, and even death if administered into the human body through injectable medications, intravenous solutions or implantable medical devices. Testing helps manufacturers to effectively eliminate contaminated products from the market.

Endotoxin refers to a toxic substance that is present in the outer membrane of gram-negative bacteria. From the chemical point of view, endotoxins are lipopolysaccharides (LPS) containing lipid A moiety that produces a toxic effect and a polysaccharide chain. Endotoxins are known to incite strong responses from the body’s immune system when they circulate in the bloodstream or infiltrate tissues and can cause fever, inflammation, septic shock, and sometimes death.

Endotoxin testing becomes mandatory during manufacturing and quality control of products, which come in contact with blood circulation or are intravenously administered, be it a pharmaceutical product, a medical device, or a biologic product, etc. Products like dialysis solutions, surgical implants, as well as other sterile medical products, must also guarantee that their endotoxin levels do not go over a particular threshold.

One of the most common methods employed in the quantification of endotoxins in a particular sample is through the use of the turbidimetric method. In this test, the endotoxins cause a reaction, which leads to turbidity or cloudiness of the solution. The level of turbidity increases with the concentration of endotoxin and is determined through spectrophotometer value.

Endotoxin contamination is never acceptable in injectable medications due to possible severe adverse effects in patients. Endotoxins are capable of eliciting fever, chills, septic shock, as well as other fatal consequences if injected into the bloodstream. Quality control acceptance and regulatory compliance can only be accomplished when injectable medications are free from endotoxin.

The LAL test detects the presence of bacterial endotoxins by using the blood cells of the horseshoe crab, which clots in the presence of endotoxins.

Manufacturers of pharmaceuticals, biologics, and medical devices comply with this standard.

Samples are prepared by dissolving or suspending the test article in an appropriate solvent to ensure that the solution is endotoxin-free.

The Chromogenic method uses a color change to detect and quantify endotoxins, providing highly sensitive and quantitative results.

While USP 85 tests for endotoxins, USP 71 tests the sterility of the products.

Yes, this standard is harmonized with the European Pharmacopeia (EP) to ensure consistency across global regulatory frameworks.

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