Hayden Skalski - Veolia Water Technologies & Solutions, Sievers Instruments
In recent years, there have been major improvements and advancements in pharmaceutical technology with regards to quality control testing. One test which falls under this category is the Limulus Amebocyte Lysate (LAL) test which detects bacterial endotoxins, a harmful pyrogen, in water and final drug products. Traditional photometric LAL tests – which include the 96-well microplate assay for kinetic turbidimetric and kinetic chromogenic tests – are time consuming and require significant hands-on time. Now with the use of microfluidics, liquid handling and hands-on time can be significantly reduced, alongside a reduction in LAL and sample volumes while maintaining compliance.
How much lysate is needed for compliant assays?
Traditional LAL tests use 96-well microplates which have a specific shape for each well and path length for absorbance. The path length of a 96-well microplate varies slightly depending on the manufacturer or vendor. This is because the well on a flat bottom 96-well microplate is often a truncated cone, made for easy release from the molding.1
Because of the well depth and shape, LAL manufacturers specify how much sample volume and LAL reagent are needed in each well to maintain a 1:1 ratio for the detection of endotoxins. Kinetic endotoxin assays rely on an inverse relationship between the change in optical density (OD) and endotoxin concentration. OD is the “measure of the amount of light absorbed by a suspension or a solution of an organic molecule at a specific wavelength, as measured by a spectrophotometer”.2
The ratio of sample to lysate (1:1) is very important. Most LAL manufacturers recommend using 100 µL of sample to 100 µL of LAL for 96-well microplate readers and tube readers. There are some lysates that utilize less than 100 µL of sample and lysate, and these are typically run in microplates that have smaller wells. However, the 1:1 ratio is still maintained. This 1:1 ratio has been proven over years to accurately deliver results for BET. Using 100 µL of sample and lysate is not a regulatory or pharmacopeia requirement. Rather, it is a recommendation on how much volume to use to meet the 1:1 ratio given the well size in a microplate or tube reader for the desired path length and absorbance to reach optical density.
Microfluidics enables accurate, reliable, and compliant endotoxin testing using smaller reaction volumes
New innovations for LAL testing have emerged in recent years such as the Sievers Eclipse BET Platform. The Eclipse utilizes microfluidic technology and centripetal force to accurately deliver small volumes of liquid to the specific segments on the microplate, thus drastically reducing the amount of reagent needed and simplifying the setup. Microfluidics has been a thriving research field for over 30 years due to its abilities to meet demands for chemical and biological analyses that are accurate, cost-effective, reliable, and sensitive – which is exactly what is needed in BET assays.
The Eclipse microplate has a standard curve embedded in five segments, and each standard has its own negative control. There are 21 sample segments that allow a user to run up to 21 samples on one plate, similar to a 96-well microplate reader. Each of the sample segments contain endotoxin for the Positive Product Control. Because microfluidics requires smaller reaction volumes but does not alter the biochemistry, the Eclipse microfluidic technology is designed to use less lysate than typical methods, while maintaining full compliance. The optical density for the Eclipse was calculated using Beer’s Law. Beer’s Law, also known as Beer-Lambert’s Law, states that “the concentration of a chemical solution is directly proportional to its absorption of light.”3 Due to the nature of the microfluidics, including the fluidic pathway and sample/lysate chamber, the Eclipse only requires 1mL of lysate and ensures a 1:1 ratio of sample to lysate for each assay.
Ensuring equivalent performance of the Sievers Eclipse to traditional microplate readers
Extensive testing was performed throughout the development of the Eclipse to ensure equivalent performance to a 96-well microplate reader. The United States Pharmacopeia outlines in chapter <1225> the parameters to look at when validating an analytical method. The performance of the Eclipse system was evaluated by measuring different parameters outlined in USP <1225>.
Testing was performed with multiple LAL lots from multiple vendors over multiple years. In addition to LAL Reagent Water, drug product samples were run on both the Eclipse and a 96-well microplate reader. Data showed that the performance of the Eclipse was equivalent to the traditional microplate reader. In conclusion, the Eclipse uses a compendial kinetic chromogenic lysate to perform a compliant Bacterial Endotoxin Test. Validation data according to USP <1225>shows that the Eclipse is equivalent to the 96-well microplate reader.
References
- Sievers Analytical Instruments. Determining equivalent onset optical density (OD) values on the Sievers Eclipse BET platform.
- McCullough, Karen Zink. The Bacterial Endotoxins Test: A Practical Approach. DHI Publishing, LLC, 2011.
- Helmenstine, Anne Marie. Beer’s Law Definition and Equation. ThoughtCo, 2019. https:// www.thoughtco.com/beers-law-definition-and-equation-608172
Author Biography
Hayden Skalski is the Lead Life Sciences Product Application Specialist for the Sievers Instruments product line, specializing in bacterial endotoxins testing (BET). Hayden has over 8 years of experience in the pharmaceutical industry and Quality Control Microbiology and has presented on numerous topics surrounding endotoxin testing. Previously, Hayden held roles at Charles River Laboratories, Regeneron and Novartis, validating and executing method development protocols for endotoxin testing, providing customer support, troubleshooting and supporting high-volume product testing. Hayden has a B.S. from the University at Albany (SUNY) in Biology.
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