Endotoxin testing is critical in industries such as biopharmaceuticals and medical devices to ensure product safety and reliability.
Endotoxins, which are lipopolysaccharides (LPS) found in Gram-negative bacteria's cell walls, can induce severe reactions such as fever, shock, and even death if they penetrate the human body. Therefore, precise and standardized endotoxin testing is critical.
The recombinant Factor C (rFC) endotoxin assay has grown in popularity in recent years, thanks to its great sensitivity and accuracy. This article will cover the essential aspects for endotoxin testing, which will help producers in the biopharma and medical device industries maintain safety and quality.
1. Endotoxin characteristics and detection principle
Endotoxins are mostly composed of lipopolysaccharides (LPS), which are found in the outer membranes of Gram-negative bacteria, including E. coli and Salmonella. LPS is composed of three parts: lipid A, a core oligosaccharide, and an O-antigen polysaccharide.
The lipid A component is hydrophobic, but the core and O-antigen parts are negatively charged. These qualities enable endotoxins to adhere to a variety of surfaces, including unprotected glass, metals, plastics, and even biological materials.
Endotoxins are difficult to neutralize using standard disinfectants such as alcohol or phenol, and they can resist severe temperatures and pH levels.
When they penetrate the human body, they might cause adverse inflammatory reactions. This is why endotoxin testing is so important in the pharmaceutical sector to ensure the safety of medications and medical devices.
The rFC endotoxin assay activates Factor C when it comes into contact with endotoxins. This contact causes a luminous response, with the intensity of the fluorescence indicating the level of endotoxins present. This procedure is quick, accurate, and very specific.
2. Preparation for endotoxin testing: Key steps for reliable results
In practical testing, the endotoxin content in samples may be either too high or too low, surpassing the detection method's linear range. If the concentration is too high, signal saturation can occur, making it hard to identify the true endotoxin level.
If the signal is too low, it may be feeble and sensitive to background noise interference, reducing the accuracy of the results. In addition, matrix effects, which are interferences generated by other components in the sample, can alter the test results.
Proper dilution brings the endotoxin concentration within the assay's linear range while also reducing matrix effects, improving the test results' accuracy and reliability.
- Environmental requirements
To avoid contamination from external endotoxins, testing should be performed in a clean environment, preferably at a dedicated clean workstation.
A clean workplace requires regular cleaning, disinfection, and air purification. To eliminate outside disturbance, keep the lab temperature between 18 and 25 °C and minimize unnecessary movement during testing.
- Instrument and reagent preparation
- Instruments: Ensure all equipment, including fluorescence detectors, thermostats, and pipettes, are calibrated and working properly. Before using the instruments, ensure that they are in good working order, with no errors or malfunctions.
- Reagents: Reagents and standard endotoxin solutions should be stored at 2-8 °C, according to specifications. To ensure the purity and effectiveness of your solutions, always use endotoxin-free water.
- Consumables: To avoid outsourcing contamination, prepare samples with endotoxin-free pipette tips and glassware. Make sure to thoroughly vortex the samples to ensure consistent mixing.
3. Key considerations during the endotoxin testing process
During sample collection, make sure that all sampling instruments are sterile and free of endotoxins. To avoid outsourcing contamination, use single-use sterile sampling equipment and adhere to the recommended sampling techniques.
Handling varies depending on the type of sample. For liquid samples such as injections, utilize endotoxin-free diluents and adhere to proper dilution ratios. Solid samples must be thoroughly dissolved without adding endotoxin contamination. Test samples as soon as possible to prevent variations in endotoxin levels.
When producing the rFC endotoxin assay reagents, carefully follow the manufacturer's instructions. To ensure accuracy in standard endotoxin solutions, employ stepwise dilution. Make sure your pipettes are calibrated for precise liquid handling.
When reconstituting reagents, gently agitate the solution (avoid violent shaking) and adhere to the recommended temperature and time restrictions. Use the reconstituted reagents right away to limit the possibility of outsourcing contamination.
- Sample addition and reaction
Add the samples in the correct order to avoid confusion. Separating samples, standards, and blanks is the best way to reduce outsourcing contamination. After adding the samples, cover the plate and set the thermostat to 37 °C for the reaction. To keep the response stable, avoid opening the thermostat.
4. Results observation and analysis in endotoxin testing
The rFC assay employs fluorescence signals to evaluate endotoxin levels. To maintain signal accuracy and stability during the observation process, verify that the instrument settings are right and that the test is carried out under adequate conditions.
- Result analysis and reporting
Pharmacopeia standards state that the interpretation of endotoxin test results must meet three criteria:
- The standard curve's correlation coefficient (R²) should be at least 0.98.
- The negative control's fluorescence signal should be lower than the lowest concentration point on the standard curve.
- Spiked samples should have a recovery rate of 50-200 %.
Only when both of the first and second conditions are met can the standard curve be regarded valid for determining endotoxin content in the sample and total content after spiking. Moreover, if the recovery rate of the spiked samples matches the third requirement, the endotoxin test findings may be regarded acceptable.
If any of the conditions are not met, the exam has to be redone.
About ACROBiosystems
ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.
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