Bacterial Endotoxin vs. Bioburden Testing
What is an endotoxin?
The “endo” in endotoxin refers to something that is within. The “toxin” component of endotoxin relates to something poisonous. Endotoxins can be deadly to humans and come from gram-negative bacteria’s cell walls. Endotoxins are molecules with fat components and complex sugar structures (also known as polysaccharides). For this reason, endotoxins are also known in scientific literature as lipopolysaccharides (LPS). Endotoxins are also considered pyrogens because they trigger the innate immune system and produce fever when released within the human body. This article covers bacterial endotoxin vs. bioburden testing, USP bioburden testing, USP bacterial endotoxin tests, and how to define bioburden vs. endotoxins.
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 from a particular sample. The higher the concentration of viable microorganisms 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 human immune system’s effort. Note that how you define bioburden vs. endotoxins is very different.
What are the differences between endotoxins and bioburden?
As mentioned above, how you define bioburden vs. endotoxins is very different. Bioburden is a broader term than endotoxin. Bioburden refers to all microorganisms (fungi, bacteria, etc.), whereas endotoxins are explicitly related to gram-negative bacteria. Bioburden relates to a quantitative concentration. This quantitative concentration is often a viable cell count expressed as colony-forming units. In contrast, the word endotoxin has no quantitative component. Endotoxin is a descriptive term used to define a piece of gram-negative bacteria that is harmful to humans.
As mentioned earlier, bioburden refers to a viable cell count. Bioburden quantifies viable microorganisms because microorganisms are of infectious concern while alive. In contrast, endotoxins are a non-living component of gram-negative bacteria. Furthermore, endotoxins are released when gram-negative bacteria have been dissolved or destroyed. In other words, gram-negative bacteria release endotoxins when they are dead.
What are the differences between bioburden testing and bacterial endotoxin testing?
USP bioburden testing measures the viable organisms present in a given sample expressed as a total viable count. This total viable count gives a value to the microbial contamination level upon or within a particular product. In contrast, bacterial endotoxin testing measures the presence and quantity of endotoxins for a specific sample. As bioburden and bacterial endotoxin tests measure and identify different things, these tests use separate assays to evaluate bioburden and endotoxins, respectively. Bioburden testing follows the methods outlined in USP 60, USP 61, and USP 62. Bacterial endotoxin testing uses the methodologies outlined in USP 85. For endotoxin limits, please visit our articles specific to medical devices, combination products, and drugs or biologics.
Bioburden testing can identify what types of live microorganisms are present in a sample from a product, package, or manufacturing environment. However, bioburden testing cannot detect bacterial endotoxins, as endotoxins are not live cells. Thus, if a bioburden test comes back positive for gram-negative bacteria, the bioburden test indicates that bacterial endotoxins could be present. However, bioburden testing will never confirm the bacterial endotoxins are present or the level of bacterial endotoxin contamination of a particular sample. Furthermore, even if a bioburden test came back negative for gram-negative bacteria, endotoxins from dead gram-negative bacteria could still be present in a sample.
Why are both bioburden testing and bacterial endotoxin 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 contamination sources, regular bioburden testing supports the long-term control of manufacturing sites.
Bacterial endotoxin testing is just as crucial as bioburden testing, especially for medical products or devices inserted into the human body. Suppose bacterial endotoxins exist on a medical device’s surface or are present within an injectable product. In that case, the exposed patient’s innate immune system will be activated upon device or product use, causing fever, chills, and potentially death.
Overall, both bacterial endotoxin and bioburden testing are imperative for a medical device or product’s regulatory approval. These tests ensure that medical devices and products are sterile enough that patients will not be at risk of infection or pyrogenesis following device exposure or product implantation.
Ethide Labs is a contract testing organization that specializes in Bacterial Endotoxin Testing. Ethide Labs also offers Bioburden Testing, Environmental Monitoring, Sterilization Validations, Microbiology Testing, EO Residual Testing, Package Integrity Testing & Cytotoxicity Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
Galanos and M. A. Freudenberg. Bacterial endotoxins: biological properties and mechanisms of action. Mediators of Inflammation. 1993; 2(7): S11–S16.
Frank R. Noyes. Noyes’ Knee Disorders: Surgery, Rehabilitation, Clinical Outcomes. Elsevier. 2010.
United States Pharmacopeial Convention. <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests. Rockville, MD, USA. 2021. (USPC <61>)
United States Pharmacopeial Convention. <85> Bacterial Endotoxins Test. Rockville, MD, USA. 2021. (USPC <85>).
