The bacterial endotoxin test is a critical test performed to confirm that a raw material, product sample or component is below the limit for detecting bacterial endotoxin. Endotoxin can cause a pyrogenic response in patients, and testing for its presence is one of the most important quality control tests used to assess material and product quality.
Endotoxins are part of the cell wall of gram-negative bacteria and are always connected with product contamination when present. Endotoxins by nature are unaffected by sterilization processes using steam, ethylene oxide (EtO), or radiation, as well as most disinfection methods. All this to say that working with proper aseptic technique is critical for aseptic manufacturing.
Endotoxin is different from other types of bacterial contamination because endotoxin may be present on containers and in drug products, even when the bacteria are no longer there, or have been killed. Endotoxin contamination must be prevented rather than attempting to eliminate it upon detection. In patients, endotoxin can enter the bloodstream causing symptoms such as hemorrhagic shock, diarrhea, meningitis, fever, and dangerous blood pressure fluctuations.
Parenteral products require release testing at the end of filling to ensure they are free of endotoxin. But that is not enough. Aseptic processes need to establish critical process parameters (CPPs) to identify where the detection of endotoxin contamination has the potential to impact product quality. Once the aseptic process is evaluated, places throughout the process can be identified for endotoxin incursion. These places are established through a thorough risk assessment. Throughout the process -- from raw material testing to final product containers -- sampling points, testing, and limit-setting are necessary to establish and follow. This process provides a manufacturer with the appropriate risk control strategy.
Virtuosi®, powered by Quality Executive Partners (QxP), provides specific education in the application of the endotoxin test using virtual reality and substantive content for the Quality Control (QC) analyst. Bacterial endotoxin is typically detected by using a reagent known as Limulus Amoebocyte Lysate (LAL), also known as Lysate for short. Bacterial endotoxin causes a clotting reaction in lysate in a manner that is directly related to the concentration of the endotoxin. The specific concentration of endotoxin that should cause clotting of the lysate is known as the lysate sensitivity or lysate activity. One of the most important controls in the performance of the test is the standardization of the purchased lysate, to confirm its labeled sensitivity. This test essentially determines whether the lysate can indeed clot when it is exposed to concentrations of endotoxin that are at or above the labeled lysate sensitivity. The results of the clotting reaction in the test are important to the Quality Control and Quality Assurance Departments so they can determine if the materials or finished product conforms to pre-determined specifications.
Looking forward, there have been several recent developments in the industry for bacterial endotoxin testing. The United States Pharmacopeia (USP) recently updated the guidelines to include new technologies such as recombinant factor C (rFC), which is the synthetic version of limulus amoebocyte lysate, commonly harvested from a horseshoe crab. Increased use of recombinant technologies will help to eventually ensure robust supply chain for this type of reagent, especially considering the market expansion with cell and gene therapies. The rFC may be more cost-effective and more sustainable than traditionally sourced reagents. The European Pharmacopeia (EP) has also revised its bacterial endotoxin testing compendia to include the use of alternative methods, including rFC as a potential reagent, thus improving upon the traditional endotoxin detection methods such as kinetic chromogenic and turbidimetric assays.
But this does not stop here. There is ongoing research to create completely new methods of endotoxin testing, such as microfluidics and biosensors. The goal for these changes is to improve upon accuracy, effectiveness, and sustainability of the reagents and the testing process.
Quality Executive Partners (QxP) applauds the industry in these steps forward. The experts at QxP partner with firms to support excellence in manufacturing, including establishing robust endotoxin methodologies. We can help you assess your processes using our risk assessment tools to ensure endotoxin is part of your overarching contamination control strategy. In addition, the QxP education system, Virtuosi®, can strengthen your workforce and prepare your firm for new changes to already validated testing methods. Click here to connect with us.
QxP Vice President Christine Feaster is a 20+ year veteran in pharma quality assurance. Prior to joining QxP, Christine was a vice president of U.S. Pharmacopeia.
