In the pharmaceutical and medical industries, guaranteeing product safety is of utmost importance, especially for products that are usually injected or come in contact with the body. Controlling bacterial endotoxins is one significant aspect that ensures this safety since they are toxic substances produced by Gram-negative bacteria and can lead to serious complications in patients, such as fever and inflammation. The USP 85 compliance defines the requirements for bacterial endotoxin testing (BET) to make sure that such products are safe for human consumption. It encompasses a variety of things, such as injectable drugs, biologics, and some medical devices, among others. 

Understanding USP 85: Bacterial Endotoxins Test (BET)

The USP 85 compliance describes the bacterial endotoxins test, BET, which plays an integral role in the detection and determination of endotoxins in pharmaceutical products. These are toxic components in the outer membrane of Gram-negative bacteria and may induce severe adverse reactions if they are introduced into the bloodstream or cerebrospinal fluid.

Purpose and Scope of USP 85 test

USP 85 compliance serves two main purposes:

• Patient’s Protection: The main objective is to protect patients from the detrimental effects of bacterial endotoxins. Compliance ensures that products, such as injectables, vaccines, and medical devices, will not carry dangerous levels of endotoxins that may lead to adverse health outcomes.

• Compliance with Regulation: The regulatory agencies enforce manufacturers to adhere to this standard so that they can make sure that parenteral products are not contaminated by any kind of endotoxin.

Products Tested as per  USP 85 Testing Protocol

USP 85 testing applies to a wide range of products, including:

Injectable pharmaceuticals: Solutions, suspensions, and emulsions meant for injection.

Biologics: Vaccines, therapeutic proteins, and monoclonal antibodies.

Medical devices: Devices that come in contact with sterile body sites, such as catheters, implants, and syringes.

Methods of Endotoxin Detection Under USP 85

USP 85 test specifies the following methods:

1. Gel-Clot Method

The gel-clot technique is one of the most commonly used methods for endotoxin testing. The gel-clot method’s principle is based on endotoxins’ ability to induce clotting cascade in LAL reagent. Upon the addition of endotoxins in a sample, it reacts with the LAL reagent and forms a gel-like clot

Advantages:

• It is very simple and inexpensive.

• High sensitivity to endotoxins.

Limitations:

• Test results might be subjected to variable interpretation.

• It can be time-consuming compared to more automated methods.

2. Turbidimetric LAL Assay:

The turbidimetric Limulus Amebocyte Lysate (LAL) assay is one of the key methods used in the bacterial endotoxin test (BET) as outlined in USP General Chapter <85>. This assay determines the increase in turbidity or cloudiness of a solution caused by the reaction of endotoxins with the Limulus Amebocyte Lysate. The presence of endotoxins initiates a cascading reaction (the product of this cascade is a clotting protein). This clotting increases turbidity. The increase in turbidity is directly proportional to the concentration of endotoxins present in the sample.

Advantages:

• Quantitative measurement.

• It can be automated for high-throughput testing.

Limitations:

• Requires specialised equipment.

• It may be affected by interfering substances in the sample.

3. Chromogenic Method

This technique is based on the colorimetric reaction, in which the endotoxins will catalyze the release of a chromophore from a synthetic substrate that can be measured quantitatively through spectrophotometry. The intensity of color change is directly proportional to the concentration of endotoxins present in the sample.

Advantages:

• Highly sensitive and quantitative.

• Automated systems allow for rapid results.

Limitations:

• Requires specialized equipment and reagents.

• Can be affected by sample matrix interference.

Steps for testing the presence of endotoxins to ensure USP 85 Compliance

1. Sample Preparation and Handling

Manufacturers must ensure that samples used in testing are truly representative of the final product offered at markets. Adequate handling of samples aids in avoiding contamination, degradation, or loss of endotoxic agents. Sterile containers are recommended for sampling.

2. Validation of Test Methods

Before carrying out regular tests, manufacturers have to authenticate their selected methods for detecting any endotoxic agents specific to their products in order to meet USP 85 compliance. It involves proving that a given test method is appropriate in terms of specificity, precision, and accuracy among others.

For some products like biologics and complex formulations, inhibition/enhancement testing might be required to confirm if there will be no interference from the sample matrix during analysis.

3. Routine Monitoring and Testing

After validating the test method, manufacturers need to carry out routine endotoxin testing at different stages of production. This may include:

• Testing of raw materials: Assuring that none of the incoming materials contain endotoxin.

• In-process testing: Measuring endotoxin levels during production to notice any early signs of contamination.

• Final product testing: Confirming whether or not the finished product meets the required endotoxin limits before it can be released into the market.

4. Compliance with Regulatory Limits

For each product type, there is a certain limit that has been set by regulatory authorities depending on the route of administration and dosage. The limit for injectable drugs is more stringent compared to that of topical products. The products have to meet the limits of the endotoxins every time they leave the manufacturing site.

Challenges  and considerations for USP 85 Compliance

  Product Matrices Interference

• Endotoxin detection methods can be interfered with by some products such as biologics and complex formulations. Additional validation steps like dilution or inhibition/enhancement testing may also be necessary to achieve accurate results.

 Calibration and Maintenance of Equipment

• Specialized equipment including spectrophotometers and turbidimeters are required for endotoxin testing; thus they should be correctly calibrated and maintained. This equipment should go through regular calibration and maintenance to avoid inaccurate or unreliable results.

Continuous Personnel Training

• It is important to properly train personnel who are responsible for endotoxin testing to handle samples, operate equipment, and interpret results. Regularized training ensures that they remain updated with the latest USP 85 test procedures and regulatory standards.

Conclusion

Testing for USP 85 compliance is important to ensure the safety, efficacy, and regulatory compliance of pharmaceutical products, biologics, and medical devices. This test detects and quantifies bacterial endotoxins to help manufacturers ensure that patients are not exposed to contaminants. Whether by gel-clot, turbidimetric, or chromogenic methods, adherence to USP 85 standards is important for maintaining product quality and meeting regulatory standards.

At MIS we offer comprehensive testing validation services for your products including USP85 (endotoxin testing) to support your efforts in ensuring regulatory compliance and product excellence. Contact us today to learn more about USP 85 testing services.