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Bioburden vs. Bacterial Endotoxin Testing: Exploring Methods, Differences, and Laboratory Practices in Medical Devices

Bioburden and bacterial endotoxin testing are two important concepts in the field of microbiology relating to pharmaceutical and medical device manufacturing. Understanding their attributes and differences is crucial for ensuring the safety and quality of medical devices, pharmaceutical products, and biological materials.

These two tests evaluate two different quality attributes of a pharmaceutical product or medical device related to the device’s final cleaning and handling processes. 

  • Bioburden testing is a quantitative assay, aiming to evaluate the number and the species of living microorganisms present on a medical device before sterilization.
  • Bacterial Endotoxin testing is a quantitative assay, which assesses the level of bacterial endotoxin present on the device.

Bioburden Testing

Unlike pharmaceuticals (liquid or powder), a medical device is not a product that can be dissolved, diluted, or divided. To analyze bioburden on a medical device, the maximum quantity of organisms (bacterial or fungal) needs to be removed from the medical device without killing them. To do so, a medical device needs to be immersed in (or filled) with a neutral eluent able to separate microorganisms from an inorganic surface without causing microbial lethality or promoting growth. Next, the medical devices are submitted to successive treatments (shaking, ultrasonic waves, etc.) to remove the microorganisms. Several methods can be used dependent on the type of tested product. The eluent can be directly included in a solid growth medium, but the preferred method is to filter the eluent then transfer the filter membrane to a solid growth media (agar) and incubate at specified conditions for detection of the microorganisms.

When screening, the types of microorganism that can encountered as routine bioburden can be differentiated (aerobic, anaerobic, yeast/mold, etc.). There may also be a need to grow the microorganism in a different culture condition, where the device’s eluent can be split, filtered, and incubated in different conditions. The obtained results need to be corrected/adjusted to estimate the total number of organisms initially present on the device by a validated correction factor.

Prior to performing routine bioburden testing, the extraction method must be validated and consideration taken that it is never possible to remove 100% of microorganisms from a medical device, statistically speaking. There are several steps necessary to validate this method:

  1. Validate the Efficacy of the Extraction: Several strategies could be used. The most classic and recommended is to inoculate sterile devices with a known amount of a specific organism (typically a spore suspension of Bacillus atrophaeus or Geobacillus stearothermophilus). Then the quantity of recovered organisms is compared against the quantity enumerated in the inoculum to determine the percentage of recovery and calculate a correction factor to apply to routine bioburden testing results. The aim is to be as close as possible to the real quantity of the living bioburden present on a device. Other methods can be used, such as repetitive extraction of non-sterile devices using the natural bioburden of the product to test as recommended in ISO 11737-1.
  2. Evaluate the In Vitro Conditions Used in Routine Testing (Bioburden Suitability Testing/Screening for Inhibitory Substances): The presence of the device or the physical extraction of organisms causes inhibition (or growth promotion) of the bioburden organisms during the treatment, leading to a potential underestimation of the real quantity of living microorganisms present on the tested product. A variety of representative vegetative microorganisms are used to inoculate the eluent. By adding these germs to the eluent, the method of extraction or the device materials can be evaluated to see if it affects the number of organisms recovered compared to the known inoculated quantity.

Controlling bioburden is crucial to prevent microbial contamination, and potential harm to patients or users. It is achieved through various means, including proper facility design, cleaning and disinfection procedures, personnel training, and sterilization techniques. Regular monitoring of bioburden levels is essential to ensure the effectiveness of control measures and to maintain product quality and safety.

Bacterial Endotoxin Testing

Endotoxin, also known as lipopolysaccharide (LPS), is a component of the outer membrane of Gram-negative bacteria. It is a potent pyrogen, capable of inducing fever and inflammatory responses in humans and animals. Endotoxin contamination is a significant concern in pharmaceutical manufacturing and implantable medical devices, as it can cause severe adverse reactions in patients.

The primary source of endotoxin is Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Salmonella species. These bacteria can contaminate raw materials, water, equipment, and manufacturing environments. Endotoxin can be introduced during various stages of production, including raw material processing, formulation, and final cleaning processes.

Endotoxin testing is performed to quantify the presence of endotoxin in pharmaceutical products and medical devices. The most commonly used method is the Limulus Amebocyte Lysate (LAL) assay, which utilizes the clotting reaction of horseshoe crab blood in the presence of endotoxin. The results are expressed in endotoxin units (EU) per unit of the product or material.

Strict limits for endotoxin levels are established based on regulatory guidelines and product-specific considerations. These limits are often expressed in EU per milliliter (EU/mL) for parenteral products or EU per device for medical devices. To ensure compliance, manufacturers implement rigorous process control measures, including proper facility design, water purification systems, cleaning and disinfection procedures, and validation of the manufacturing processes.

Routine testing for endotoxin is essential to ensure the safety and quality of pharmaceutical products as well. It helps identify potential sources of contamination, evaluate the effectiveness of control measures, and prevent adverse reactions in patients. Endotoxin testing is particularly critical for products administered intravenously, directly into the bloodstream or permanently implanted.

Bacterial Endotoxin Testing Methods

  1. Limulus Amebocyte Lysate (LAL) Assay:
    • Description: Uses blood cells from horseshoe crabs that clot in the presence of endotoxins. There are three types of LAL assays:
      • Gel-Clot Method: Endotoxins cause the formation of a gel clot.
      • Turbidimetric Method: Measures the increase in turbidity caused by endotoxins.
      • Chromogenic Method: Measures color change resulting from endotoxin interaction.
    • Pros: Highly sensitive and specific for endotoxins.
    • Cons: Requires careful handling and preparation.
  2. Recombinant Factor C (rFC) Assay:
    • Description: Uses a genetically engineered version of the Factor C protein from horseshoe crabs, which reacts with endotoxins.
      • Pros: Avoids the use of animal-derived products and is highly specific.
      • Cons: Relatively new and may require validation for some applications.

These methods ensure the safety and quality of products by accurately detecting and quantifying microbial contamination and endotoxins.

What is the Difference Between Bioburden and Bacterial Endotoxin Testing?

While both bioburden and endotoxin are related to microbial contamination, they differ in several aspects. Bioburden focuses on the total microbial load, including viable microorganisms. Bioburden testing provides a broader assessment of microbial contamination, considering all types of microorganisms, including bacteria and fungi. On the other hand, bacterial endotoxin testing specifically targets the presence of endotoxin from Gram-negative bacteria, which is a potent pyrogen and can cause severe adverse reactions in humans.

Both tests play vital roles in pharmaceutical manufacturing and medical device production. Controlling bioburden levels is essential to prevent microbial contamination, product spoilage, and potential harm to patients or users.  Regular monitoring of bioburden helps ensure the effectiveness of control measures and maintain product quality and safety.

Bacterial endotoxin testing is critical for parenteral products/devices and implantable medical devices. Regular testing helps identify potential sources of contamination, evaluate the effectiveness of control measures, and prevent adverse reactions.

Both bioburden and endotoxin testing require manufacturers to implement robust quality control systems, including proper facility design, cleaning and disinfection procedures, personnel training, and sterilization techniques. Compliance with regulatory guidelines and product-specific considerations is essential to ensure the safety and efficacy of pharmaceutical products and medical devices.

AttributeBioburdenEndotoxin
DefinitionThe total number of viable microorganisms present in or on a product or surfaceToxic substances found in the outer membrane of certain gram-negative bacteria
MeasurementUsually expressed as colony-forming units (CFU) per unitMeasured in endotoxin units (EU) per unit
OriginCan come from various sources, including air, water, raw materials, and personnelProduced by certain bacteria, primarily found in the gastrointestinal tract and water
PresenceBioburden can be present on both living and non-living surfacesEndotoxins are only found in the outer membrane of gram-negative bacteria
Health ImpactHigh bioburden can indicate poor hygiene and increase the risk of contamination and infectionEndotoxins can cause fever, inflammation, septic shock, and other adverse reactions in humans
Testing MethodsBioburden testing involves microbial enumeration through culture-based methods or molecular techniquesEndotoxin testing is typically performed using the Limulus Amebocyte Lysate (LAL) assay

Table: Bioburden vs. Endotoxin Testing

Conclusion

Bioburden and endotoxin are two important concepts in microbiology, pharmaceutical, and medical device manufacturing. While bioburden focuses on the total microbial load, endotoxin specifically targets the presence of endotoxin from Gram-negative bacteria. Bioburden testing provides a broader assessment of microbial contamination, while bacterial endotoxin testing is specific to endotoxin, which can cause severe adverse reactions in humans. Both testing methods have established limits and require manufacturers to implement robust control measures to ensure product safety and quality. Regular monitoring of bioburden and endotoxin levels is crucial to maintain compliance and prevent harm to patients or users.

Frequently Asked Questions (FAQ)

Do I need to perform bioburden or bacterial endotoxin testing?

Depending on the type of sterilized device that you are manufacturing, you may need to do both.  As bioburden is conducted as part of the initial validation process, then is done on a scheduled frequency (i.e. quarterly) to monitor and trend the data as part of a quality control function.  While bacterial endotoxin testing is required on a lot-by-lot basis for devices labeled as “non-pyrogenic”.

How long does bioburden and/or bacterial endotoxin testing take on average?

Bioburden testing typically takes 7-10 days to complete at a laboratory, due to the various incubation durations to allow the recovered bioburden organisms to proliferate on the growth media.

Bacterial endotoxin testing does not require as much time as the bioburden testing, as it is a kinetic chemical assay for detection of the levels of endotoxin present in the device extract.  This can be conducted between 1 to 3 days, depending on the testing queue at the laboratory.

What does the process look like to initiate bioburden and/or bacterial endotoxin testing?

Bioburden testing is conducted on devices (typically 10 samples) that have been packaged using routine manufacturing and handling process, but not sterilized.  The non-sterile devices would then be sent to the laboratory to then be processed.

Bacterial endotoxin testing can be conducted on either sterile or non-sterile samples (since the current terminal sterilization processes have not been shown to actively reduce endotoxin levels) but it is typically conducted on sterilized devices as part of the lot release process.  The sterile samples are sent to the laboratory to extract and quantify the levels of bacterial endotoxin present on the devices and report if it was within the acceptable limits of the device category for which it belongs.


Ed Arscott, BS

Ed Arscott, BS

Ed has a long history with NAMSA, starting in 1987 and progressing to the role of Manager of Microbiology and In vitro Toxicology. After a 14-year stint with Depuy/J&J, he returned to ԰in 2013 as a Senior Product Development Specialist. In this role, Ed provides consultation on various aspects of medical device development, including terminal sterilization methods, packaging shelf-life studies, and cleaning efficacy validation. He is also a subject matter expert for auditing external contract laboratories and sterilization vendors. Ed's recent projects include coordinating the design and validation of a new cleanroom and conducting an epidemiological risk assessment for a reusable oral medical device.