Bioburden vs. Pyrogens In Biocompatibility Testing For Cosmetics
What is bioburden?
The “bio” in bioburden refers to live biological organisms, and the “burden” in bioburden refers to the concentration of the viable biological organisms. Thus, bioburden is the concentration or quantity of microorganisms in a given area or from a particular sample. The higher the concentration of viable organisms on a device or product, the higher the burden is to kill those organisms, whether it is killing the organisms through sterilization procedures or killing the organisms through the effort of the human immune system.
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 found 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 normal 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 toll-like receptors (TLRs) to develop a fever. Clinically, the fever produced by endogenous cytokines is indistinguishable from fever produced by exogenous pyrogens such as endotoxins.
Why are endotoxins harmful, and how do endotoxins cause pyrogenesis?
The lipopolysaccharide (LPS) components that endotoxins are composed of are foreign to the human body. Thus, our immune system identifies LPS as something foreign that it should get rid of quickly. Compare our body to a house and our immune system to the inhabitants of a home. In this situation, the innate immune system treats LPS like an uninvited guest (such as a burglar or a rodent). Once triggered, the innate immune system does its best to get the uninvited guest (LPS) out of the house (the human body). The activation of the innate immune system by endotoxins results in a fever (pyrogenesis). If endotoxins run unchecked within the human body, an individual can become septic and die. Thus, appropriate pyrogenicity testing (also known as a bacterial endotoxin test) is vital to patient safety for certain cosmetic products. Endotoxin testing can be performed on raw materials used to create cosmetics and the final formulation.
How do pyrogens, endotoxins, and microbes relate?
The most ubiquitous source of pyrogens for medical products and medical devices is endotoxins. Endotoxins come from the cell wall of microbes, specifically the cell wall of gram-negative bacteria. Endotoxins are not gram-negative bacteria themselves. However, the concentration of gram-negative microbes in a manufacturing environment impacts the endotoxin concentration of a medical product or device. Thus, the better an environment reduces microbial burden (particularly gram-negative microbial load), the better protected a manufactured medical device or product is from high levels of endotoxin contamination.
What is a bioburden test?
Bioburden testing measures the viable organisms present in a sample. The total viable organisms present is expressed as a total viable count. This total viable count (expressed as CFU’s) provides a value for the microbial contamination level within a particular sample. Combined bioburden and microbiology testing can identify what types of live microorganisms are present in a sample from a product, package, or manufacturing environment. Though bioburden testing may be referred to synonymously with microbiology testing, microbiology testing is what allows for the specific type of live microorganism to be identified and quantified. Bioburden testing follows the methods outlined in USP 60, USP 61, and USP 62.
What is a pyrogenicity test?
A bacterial endotoxin test (BET) uses an assay known as the Limulus Amoebocyte Lysate (LAL) test. Bacterial endotoxin testing is an in-vitro pyrogenicity test. Furthermore, a BET is the most common, easiest, and cheapest pyrogenicity test for medical devices and products. BET testing is separate from rabbit pyrogen testing, which is rarely needed for regulatory testing. LAL is an extract of blood cells from the Atlantic horseshoe crab. LAL detects the LPS of the cell wall of gram-negative bacteria, even if these bacteria are dead. LAL detects LPS through clotting and gelling in the presence of LPS, allowing for precise calculations to be made as to the concentration of endotoxins in a sample.
Why are bioburden testing and pyrogenicity testing important?
Bioburden testing is an important quality control step that detects the level of contamination of a product at any stage, from initial product manufacture to final distribution. As microorganisms exist on every surface (including our body), bioburden can be accidentally introduced during the manufacturing or packaging process in many ways. Some of the most common examples are contamination through the raw materials used, technicians, tubing/piping used to transfer product between development stages in a process, or the manufacturing environment itself. With such abundant sources of contamination, regular bioburden testing supports the long-term control of manufacturing sites.
Pyrogenicity testing is an important quality control step that detects the level of endotoxin contamination at any stage, from initial product manufacture to final distribution. As endotoxins are the most prevalent pyrogens and microorganisms exist on every surface (including our body), bacterial endotoxins can be accidentally introduced during the manufacturing or packaging process in many ways. Some of the most common examples are contamination through the raw materials used, technicians, tubing/piping used to transfer product between development stages in a process, or the manufacturing environment itself. With such abundant sources of contamination, regular endotoxin testing supports the long-term control of manufacturing sites. Bacterial endotoxin testing is especially for cosmetic products that will be inserted into sensitive areas of the human body, such as the eyes or mouth. Cosmetics that meet bacterial endotoxin limits prevent the client’s innate immune system from being exposed to endotoxins, thus preventing the risk of fever, chills, and death with product use.
Summary
Overall, testing for pyrogens, particularly bacterial endotoxins, and bioburden are imperative safety measures for regulatory approval of cosmetic products. Pyrogenicity and bioburden testing ensure that cosmetic products are free of unwanted microbes and pyrogens so that users will not be at risk of illness following product use. Ensure you choose a contract manufacturing and testing organization that can provide appropriate biocompatibility testing for your unique product needs.
MycoScience is a contract manufacturing organization specializing in Bioburden Testing and Bacterial Endotoxin Testing. Additionally, MycoScience provides sterile syringe and vial filling for parenteral products. MycoScience also offers Bioburden Testing, Preservative Efficacy Testing, Sterilization Validations, Cleaning Validations, Accelerated Aging, Microbiology Testing, Cytotoxicity Testing, EO Residual Testing, Package Integrity Testing & Environmental Monitoring services for medical device companies, and allied industries. MycoScience is an ISO 13485 certified facility.
References
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. <60> Microbiological Examination of Nonsterile Products- Tests for Burkholderia Cepacia Complex. Rockville, MD, USA. 2021. (USPC <60>).
United States Pharmacopeial Convention. <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests. Rockville, MD, USA. 2021. (USPC <61>).
United States Pharmacopeial Convention. <62> Microbiological Examination Of Nonsterile Products: Tests For Specified Microorganisms. Rockville, MD, USA. 2021. (USP <62>).
United States Pharmacopeial Convention. <85> Bacterial Endotoxins Test. Rockville, MD, USA. 2021. (USPC <85>).
