Rubber stoppers are also a potential source of bacterial endotoxins. Due to their chemical characteristics, they are usually depyrogenated through a series of washing and rinsing cycles prior to the final steam sterilization procedure. It is important to note that the final rinse needs to be with WFI, and it is important to minimize the lapsed time between washing and sterilizing as moisture on the rubber stoppers can support microbiological growth.
Bacterial endotoxins are strongly regulated and strictly tested for in regards of parenteral drug products. USP chapter <85> titled “The Bacterial Endotoxin Test” outlines three test methods for bacterial endotoxins. The three test methods are the gel-clot method, the turbidimetric method and the chromogenic method. Although these three methods are different, they are based on the same principle which is the fact that bacterial endotoxins react with Limulus Amebocyte Lysate (LAL).
LAL is an aqueous extract of blood cells from horseshoe crab. Diagram 1 displays the overall cascade. When bacterial endotoxin reacts with Factor C it becomes activated which activates sequential enzymes within the cascade. Ultimately the proclotting enzyme turns into the clotting enzyme which causes the coagulen to turn into coagulin. Coagulin is the protein from horseshow crab blood that forms a gel-clot when reacted with bacterial endotoxin. This is the basis behind the gel-clot method. For the turbidimetric method, a turbidimetric assay is introduced within the reaction which causes turbidity if bacterial endotoxins are present. This is the basis behind the turbidimetirc method. For the chromogenic method, a chromogenic substrate is introduced to the reaction. If bacterial endotoxins are present the substrate is cleaved and a yellow color is produced. This is the basis behind the chromogenic method.
As you can see all three methods are based on the same principle which is the fact that bacterial endotoxins react with LAL; however, they differ in the last couple of steps.
Particulate cleaning and depyrogenation are critical processes that when validated reduces sub-visible particles and bacterial endotoxins to acceptable levels. There are many factors to consider when performing these procedures such as the chemical characteristic of the packaging component, cycle time of the specific process and intended use of the packaging component. These are just a few of the many considerations to take into account when packaging parenteral drug products. Yet, with a complete understanding of processes and test methods, the final drug product will not contain any contaminants that could harm the end-user.
- Gecsey, J., & Harrison, T. (2005). Sampling and preparation techniques key to success in meeting new requirements for particulate analysis in SVPs. European Journal of Parenteral & Pharmaceutical Science, 10(3), 79 82.
- General Chapter <1> Injections. USP 36/NF 31: United States Pharmacopeia. www.usp.org
- General Chapter <788> Particulate Matter in Injections. USP 36/NF 31: United States Pharmacopeia. www.usp.org
- Langille, S. E. (2013). Particulate Matter in Injectable Drug Products. PDA Journal of Pharmaceutical Science and Technology, 67(3), 186-200.
- Singh, S. K. (2013). Particulate Matter in Sterile Parenteral Products. In P. Kolhe, M. Shah, & N. Rathore (Eds.), Sterile Product Development: Formulation, Process, Quality and Regulatory Considerations (Vol. 6, Advances in the Pharmaceutical Sciences, pp. 359-409). AAPS.
- Williams, Kevin L. Endotoxins: Pyrogens, LAL Testing and Depyrogenation. 3rd ed. New York: Informa Healthcare, 2007. Print.
- Endotoxins and Their Detection With the Limulus Amebocyte Lystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, NY (1982)