How to depyrogenate sterile products by rinsing
What is depyrogenation?
Depyrogenation is a process that removes pyrogens. The most prevalent and problematic pyrogens are the bacterial endotoxins found in the outer cell walls of gram-negative bacteria. Thus, depyrogenation is a process that will either destroy or remove bacterial endotoxins. What is depyrogenation by rinsing, endotoxin removal protocols, solvent pressure regulators, solvent temperature, and more will be covered in this article.
What are pyrogens?
Pyrogens are any molecules or substances that cause a feverous reaction when they enter the human body. Endogenous pyrogens (such as the cytokine interleukin-1) are found naturally within the human body. Endogenous pyrogens create a fever-producing reaction when naturally produced by the body. Exogenous pyrogens are molecules located outside of the body, such as endotoxins from gram-negative bacteria or pyrogenic prions. For context, prions are misfolded proteins that can transmit their misfolded shape onto regular versions of the same protein. Prions are of most concern for medical products and devices that interact with brain or spinal tissues. Exogenous pyrogens either provoke endogenous pyrogen production to create a fever within the body or activate the body’s toll-like receptors (TLRs) to trigger a fever. Clinically, the fever produced by endogenous cytokines is indistinguishable from fever produced by exogenous pyrogens such as lipopolysaccharide (LPS).
Why is depyrogenation needed?
Medical devices and parenteral products must be sterile and pyrogen-free. Products can accumulate pyrogens from raw materials or other parts of the manufacturing process. The best pyrogen removal or destruction process (known as depyrogenation) depends on the product. Standard depyrogenation methods are dry heat, rinsing, and filtration.
What is depyrogenation by rinsing?
Depyrogenation by rinsing is the process of destroying bacterial endotoxins through warm pressurized water exposure.
depyrogenation by rinsing: How is the endotoxin removal protocol performed?
Rinsing is the best means to remove bacterial endotoxins on closures (such as elastomeric stoppers), medical devices, and other materials that are not compatible with dry heat depyrogenation. The depyrogenation rinsing process is completed using high-purity water solvent pressurized and at a temperature above 60°C. Multiple rinse cycles occur to remove pyrogens and ensure that the high-purity water is not contaminated with bacterial endotoxins during processing. Since rinse water purity is key to successful pyrogen removal, the rinse water quality must meet a minimum standard of less than 0.25 Endotoxin Units (EU) per milliliter. Poor water quality for rinsing may add pyrogens to medical devices and products at the same rate it removes them. Further, drying the items directly after rinsing is imperative to prevent microbial proliferation and post-rinsing endotoxin increases. Water for Injection (or other highly pure water) held for more than three or four hours below 55°C and above 8° should be considered a microbial contamination risk unless the water is sterilized. Solvent under pressure other than water, such as caustic alkali or detergents, can be used for depyrogenation by rinsing. However, these solvents temperatures need to be regulated and they may leave harmful residuals on products. Thus, if a solvent under pressure (other than water) is used for rinsing, the depyrogenation validation must prove no toxic residuals are left on depyrogenated products.
Depyrogenation by rinsing: Critical factors to define and control during endotoxin removal protocols are:
- Solvent description (including detergent description if used and the source of high-purity water)
- Solvent temperature
- Solvent pressure
- The flow rate of the solvent through the system
- Solvent recirculation (justification if used)
Validation
Validation of depyrogenation utilizes endotoxin indicators (typically a thousand endotoxin units per article) to simulate product endotoxin contamination. However, other endotoxin concentrations can be used to evaluate the depyrogenation cycle depending on: 1) the product’s historical endotoxin contamination level, 2) the number of recoverable endotoxins extracted after the product’s drying process, and 3) the “safe” level of endotoxin activity that must be attained for product use. Quantitation of endotoxin activity for depyrogenation is determined using the Limulus amoebocyte lysate (LAL) assay. Endotoxin activity is calculated before and after the rinsing depyrogenation process to demonstrate endotoxin removal effectiveness. Testing of inoculated and uninoculated indicators is performed as controls for depyrogenation validation studies.
When removing endotoxins from a product, the established endotoxin limit must not be exceeded by the sum of the endotoxin components of the active pharmaceutical ingredient (API) and any contributions by the excipients or other additives for parenteral products, pharmaceuticals, and other formulation products. For depyrogenation, the goal is often a 3-log reduction in recoverable endotoxin for the product. However, where a 3-log reduction is not required, inherent endotoxin burden and calculated endotoxin contamination risk can be used to justify depyrogenation processes that provide less than a 3-log reduction in pyrogens.
The log reduction is calculated using the following formula:
Log10 reduction = (log10 recoverable activity) − (log10 residual activity)
Summary
Overall, depyrogenation is a process that removes pyrogens. Testing for pyrogens is an imperative safety metric for regulatory approval of a medical device or product. The most prevalent and problematic pyrogens are the bacterial endotoxins found in the outer cell walls of gram-negative bacteria. Depyrogenation by rinsing is the process of removing bacterial endotoxins through exposure to warm, pressurized water. Rinsing is a common depyrogenation method for medical products and other materials that cannot undergo dry heat depyrogenation. All in all, ensure you choose a contract testing organization that can provide appropriate bacterial endotoxin testing and depyrogenation for your product needs.
Ethide Labs is a contract testing organization specializing in Bacterial Endotoxin Testing and Sterilization Validations. Ethide Labs also offers Microbiology Testing, Bioburden Testing, Sterility Testing, EO Residual Testing, Cytotoxicity Testing, Environmental Monitoring & Package Integrity Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
Charles A. Dinarello. Review: Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed. Innate Immunity. August 1, 2004.
Galanos C. and Freudenberg M. A. Bacterial endotoxins: biological properties and mechanisms of action. Mediators of Inflammation. 1993; 2(7): S11–S16.
United States Pharmacopeial Convention. <85> Bacterial Endotoxins Test. Rockville, MD, USA. 2021. (USPC <85>).
United States Pharmacopeial Convention. <1228.4> Depyrogenation By Rinsing. Rockville, MD, USA. 2021. (USPC <1228.4>).
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