One of the most meticulous assays in a quality control laboratory is the bacterial endotoxins test. With patient lives at risk, the criticality of the test is tremendous. One failure can result in costly retests, out-of-specification (OOS) investigations, and even substantial recalls. Another part of the challenge is that it is not just routine product testing that is tedious. Often, the path to arrive at routine product testing is also filled with time-consuming steps – all crucial to achieve safety and compliance, but laborious nonetheless. Many calculations and characterization studies have been developed to ensure each product is properly analyzed and determined safe for use in hopes of preventing costly failures.
Here are some of the challenges experienced on the path to routine testing:
- Calculating endotoxin limits and maximum valid dilution (MVD)
- Method suitability and product validation
- Lysate qualification
The endotoxins test has not always been what it is today. It has evolved through the years from the rabbit pyrogen test to the kinetic methods most commonly used today. One of the most common kinetic methods used in the modern laboratory is the kinetic chromogenic assay. In order to deploy the kinetic chromogenic assay, several steps and tests must be completed to ensure a product can be analyzed using this method. These steps are typically used when developing and releasing a new product, transitioning to a new LAL vendor, or switching to a new compendial method.
Calculating Endotoxin Limits and MVD
Several calculations must be completed initially to determine how to analyze a product and deem it safe for use. The endotoxin limit, maximum valid dilution (MVD), and/or a minimum valid concentration (MVC) are calculated to ensure a product does not cause an adverse effect in a patient and can be adequately analyzed in a way that adheres to regulatory requirements. USP <85>, USP <1085>, AAMI standards and other regulatory guidelines provide equations and further guidance for determining these values. These calculations can be done by hand or with the assistance of certain endotoxin software that has the capability of completing these calculations.
Once determined, these limits will be a part of the product’s lifetime so long as there are no formulation changes. One important note to remember is the sensitivity of the assay that was chosen during these calculations. If this sensitivity changes based on platform or standard curve, it is important to recalculate these values to ensure the correct MVD is being used for the next step. Employing an endotoxin platform with software that has built in calculations for endotoxin limits and MVD will save labs a significant amount of time.
Method Suitability and Product Validation
Most pharmaceutical products have been found to interfere (inhibit or enhance) to some degree with BET assays, but because of the high degree of sensitivity with LAL assays, these interferences can usually be overcome by dilution in Water for BET. After calculating the endotoxin limit and MVD, a study must be completed to determine the dilution required to overcome interference (DROI). This study, often referred to as method suitability or product characterization, analyzes the product at multiple dilutions up to and potentially including the MVD.
During this process, percent recovery of the positive product control (PPC) is closely monitored to look for any potential inhibition or enhancement that may be occurring. The percent PPC recovery is used to determine the best dilution at which to analyze the product. This study should be completed using several replicates of each dilution to show repeatability and account for any errors that may occur during the preparation of the microplate. If there is significant product-specific interference that has proven difficult to overcome by dilution in Water for BET alone, the following mitigation options are available: using an alternative lysate formulation, heat deactivation (in the presence of proteins), using a tris or endotoxin-specific buffer, or diluting with a 1 mM chelating agent (EDTA).
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Once the DROI and potential sample pre-treatment are identified, a minimum of three unique lots of the product must be tested with the same lysate vendor and method to be considered validated. This analysis of three unique lots was often tracked by the QC lab outside of the software, but some endotoxin software offers the ability to track product validation, review each lot of the validated product, and sign off on product validation. These software features can speed up the validation process by keeping results organized and easily accessible.
Lysate Qualification
Beyond a product characterization study and after a platform has been set up and validated, the lysate being used must be qualified. This is accomplished using a minimum of three endotoxin concentrations spanning the range outlined by the lysate manufacturer and is performed using at least three replicates of each concentration used. This test must be completed with each new lot of lysate to ensure it is suitable for use.
Whether completing method suitability testing, lysate lot qualification, or routine testing, the most time-consuming step is the setup of the assay microplate itself. The amount of precision and focus needed to make sure a 96-well microplate or gel-clot assay is set up without any contamination or pipetting errors is outstanding. With the traditional 96-well microplate, setup times for a full plate can extend to over an hour for even the most skilled analysts. This lengthy setup process contributes to inefficiencies even when the method suitability is biochemically basic. Testing a sample that requires repeating the suitability with another lysate vendor’s formulation can easily stretch the process to multiple days or weeks to complete, which a QC lab may not have. There are few innovative solutions available to assist QC labs in simplifying this process, but recent advances in automation enable significant time savings and simplification.
Once product validation or lysate qualification is completed, routine testing protocols can be established to streamline the daily and weekly testing needs. Some software can assist in this process by empowering analysts to create assay templates that can be quickly accessed to immediately start a test after signing in, thereby decreasing setup time and increasing sample throughput.
Overall, the bacterial endotoxins test can seem daunting with so many steps to follow, tests to complete, and assays to approve – and that is only part of the picture! Analysts must be thoroughly trained, labs must be properly set up and maintained, and compliance must be at the forefront of all processes. Using automation and software to reduce some of the pressure and burden of endotoxin testing eases the everyday strain experienced by analysts and managers within the QC lab.
Innovative technology such as microfluidic automation greatly increases sample throughput by reducing the number of pipetting steps and the amount of time required for training analysts. Ultimately, by increasing sample throughput, products can be validated more quickly with less strain on resources. Combining microfluidic technology with streamlined software enables QC labs to have products validated and lysate qualified in a timely manner, allowing for other critical lab requirements to be completed.
Author Biography
Sydney Jannetta is a Life Sciences Product Application Specialist for the Sievers line of analytical instruments at SUEZ – Water Technologies & Solutions, specializing in endotoxin testing and ultrapure water monitoring. Sydney has supported Sievers instrument customers for the last five years with expertise in TOC and endotoxin applications. She has provided method development services and feasibility testing to pharmaceutical manufacturers and has presented at over 20 national conferences. Sydney holds a Bachelor of Science degree in Chemistry from the University of Northern Colorado.