Sievers Eclipse Bacterial Endotoxins Testing (BET) Platform

Frequently Asked Questions (FAQs) for Bacterial Endotoxins Testing with Eclipse 

What is bacterial endotoxin testing?

Bacterial endotoxin testing is a critical quality control procedure used in the pharmaceutical, medical device, and biotechnology industries to detect and quantify the presence of endotoxins in various products. Endotoxins are components of the outer cell membrane of gram-negative bacteria and can cause severe immune responses in humans and animals, even in small amounts.

What are bacterial endotoxins?

Bacterial endotoxins are found in the outer membrane of the cell wall of Gram-negative bacteria. The endotoxin effect from Gram-negative bacteria is primarily due to the Lipid A component of lipopolysaccharides (LPS), which can exert pyrogenic (fever-inducing) responses that can be dangerous and even fatal when found in the bloodstream and other body fluids above certain concentrations. Because certain pharmaceuticals and medical devices enter the bloodstream, endotoxin testing is required in those industries. Due to its potent toxicity and effect on patient safety, endotoxin is globally regulated by the US FDA and international pharmacopoeia for any life sciences product that will come in contact with human and/or animal blood.

Why would you test for bacterial endotoxins in water or final products?

Within the pharmaceutical, medical device, and other life science industries, testing for endotoxins is critical for patient safety and quality control. Endotoxin tests are performed on pharmaceutical-grade water systems, including water for injection (WFI) that is used as ingredient water, and finished products such as drug, animal drug, biological, and medical device products. Pharmaceutical and medical products tested include parenteral drug products (administered through injection, such as intravenous, subcutaneous, intramuscular, and intradermal) and medical devices that have direct or indirect contact with the blood, cardiovascular system, lymphatic system, or cerebrospinal fluid. Global pharmacopoeia (USP <85>, EP 2.6.14 and JP 4.01) describe requirements for Bacterial Endotoxins Testing (BET) and endotoxin limits. FDA guidance provides recommendations for biological product, drug, and device manufacturers on current thinking concerning testing recommendations and acceptance criteria covered in compendial procedures.

How does the bacterial endotoxin test method work?

For almost 40 years, the US FDA has accepted the Limulus amoebocyte lysate (LAL) test as a finished product test for endotoxins. This is in lieu of rabbit pyrogen testing and is an in vitro assay that can detect presence and concentration of bacterial endotoxins. In LAL tests, the lysate from the blood of horseshoe crabs (Limulus Polyphemus) reacts with bacterial endotoxins to indicate the presence of endotoxins in a sample. There are three bacterial endotoxin test principle methodologies for LAL testing: gel-clot, turbidimetric, and chromogenic. In the gel-clot method, LAL and sample are mixed, followed by the operator screening for gel formation in the reaction tube. The results are from a subjective interpretation of the clot formation, and thus the gel-clot method is a qualitative test. In turbidimetric and chromogenic methodologies, samples are mixed with LAL reagent, and the color change or turbidity are measured over time. Chromogenic and turbidimetric LAL assays are quantitative and demonstrate the amount of endotoxin present. Results are calculated from a standard curve. Endotoxin limits and requirements for conducting BET are described in global pharmacopeia, USP <85>, EP 2.6.14 and JP 4.01. A new general chapter in the European Pharmacopoeia describes a method for BET using recombinant factor C (rFC) instead of the classic LAL-based method.

How does the Sievers Eclipse Platform differ from traditional 96-well microplate testing?

Traditional 96-well microplate testing requires extensive training, time to setup, and repetitive manual pipetting (up to 242 steps for 21 samples), which is time-consuming, prone to human error, and poses an ergonomic strain on analysts. The Sievers Eclipse platform utilizes microfluidic automation to drastically simplify the process, requiring fewer than 30 pipetting steps to load a full microplate. This reduces the assay setup time from roughly an hour down to about 9 minutes.

How does microfluidic automation work on the Sievers Eclipse Microplate?

The Eclipse microplate uses centrifugal force alongside a network of microchannels, metering chambers, and pneumatic chambers. As the microplate spins inside the analyzer, the system automatically and precisely distributes the samples, lysate, and water across 104 individual optical wells. This precise liquid handling ensures a perfect 1:1 sample-to-reagent ratio in every well without the need for complex robotics, securing consistent biochemistry.

How much LAL reagent is used with the Sievers Eclipse BET Platform?

With just 1 mL LAL reagent, 21 samples can be run on the Eclipse platform. By decreasing horseshoe crab (HSC) lysate use by up to 90%, the Eclipse reduces the demand of this valuable natural resource and delivers a fully compliant BET assay that the global HSC population can sustain.

Can I use animal-free recombinant reagents on the Sievers Eclipse Platform?

Yes, the Eclipse platform is fully compatible with non-animal-derived recombinant Cascade Reagents (rCR), supporting compliance with the newly introduced USP <86> guidelines. Studies comparing rCR and traditional LAL on the Eclipse have demonstrated equivalent performance for both highly purified standards and naturally occurring endotoxins (NOEs) in real-world samples.

Is the Sievers Eclipse BET Platform compliant?

Yes. The Eclipse platform uses commercially available, FDA-licensed LAL and meets all requirements of the harmonized global pharmacopoeia, USP <85>, EP 2.6.14, JP 4.01, and ChP 1143. Bacterial endotoxin testing with the Eclipse platform includes minimum three-point standard curve in duplicate using standardized endotoxin; samples and PPCs in duplicate; negative controls in duplicate; analyst and lysate lot qualification in triplicate; use of FDA licensed LAL. It also complies with 21 CFR Part 11 and data integrity guidelines.

Is the Sievers Eclipse BET Platform compliant with global regulatory guidelines?

Yes, the Sievers Eclipse platform is fully compliant with the harmonized global pharmacopoeias, including USP <85>, EP 2.6.14, JP 4.01, and ChP 1143. Furthermore, its enterprise software is designed specifically to adhere to ALCOA+ principles and 21 CFR Part 11 requirements, offering secure data transmission, customizable user permissions, and assay-specific audit trails with electronic signatures.

Are standard curves automated with the Sievers Eclipse?

Yes. Traditionally, for compliant endotoxin assays, the end user must construct at least a three-point standard curve in duplicate from a stock vial of standardized endotoxin; must have duplicate negative controls; and must run each sample in duplicate with a PPC, also in duplicate. With the Sievers Eclipse BET Platform, these steps are automated using preloaded endotoxin standards spanning up to a five-point standard curve and preloaded PPCs. Therefore, all the end user must do is load Water for BET and samples onto the plate with no additional prep work. The result is the ability to set up the assay in 9 minutes, compared to upwards of 60 minutes that other platforms require.  

Do I need to manually create standard curves and Positive Product Controls (PPCs) with the Sievers Eclipse?

No, the Eclipse microplate comes pre-loaded with Reference Standard Endotoxin (RSE) spanning up to a 5-point standard curve (e.g., 50 to 0.005 EU/mL in triplicate) and pre-deposited PPCs for every sample. Because these critical elements are embedded during manufacturing, an analyst only needs to add LAL Reagent Water or Water for BET, test samples, and 1 mL of reagent to begin the assay.

Is it difficult to train analysts and validate the Sievers Eclipse system in my lab?

No, implementation is highly streamlined for the Sievers Eclipse Endotoxin Testing System. Because microfluidics automate the most complex pipetting tasks, an analyst can be trained and certified on the Eclipse within a single day, a process that typically takes weeks or even months. System validation is equally efficient, as the platform comes with a comprehensive IQ/OQ/PQ Validation Support Package that allows the instrument to be fully validated and integrated into a lab within just a few days.

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