What is the difference between biological indicators and endotoxin indicators?
What is sterilization?
Sterilization is any process that removes, kills, or deactivates all forms of life. Sterilization is related to the term sterile, which means a complete absence of viable microorganisms or viruses that have the potential to reproduce. Sterile products that undergo sterilization are often sterilized via chemicals, heat, or radiation. Sterilization kills any microorganisms inside the products obtained during manufacturing. Sterilization usually occurs after the product is placed in its final packaging for chemical, heat, or radiation sterilization. However, sterilization by filtration tends to happen before product filling. What is depyrogenation, biological indicators (autoclave biological indicators and biological indicators for sterilization), sterilization processes that inactivate biological spores, endotoxin indicators, and more are covered in this article.
What is a biological indicator?
A biological indicator (BI) is a preparation of one or more spore-forming microorganisms that have resistance to being killed during sterilization. The spore-forming microorganisms used in biological indicators are well characterized and selected based on the tested sterilization process. Spore-forming microorganisms (gram-positive bacteria) are used as BIs because they are exponentially more difficult to kill during sterilization than non-spore-forming microbes or spore-forming microbes in an active state. Furthermore, spore-forming microbes represent a minimal subset of the actual bioburden on items to be sterilized. Thus, if a sterilization process can inactivate biological spores (the bioburden of a product represented entirely as spores), the sterilization process will be able to kill all microbes in a “worst-case” bioburden situation. The biological indicator’s ability to mimic “worst-case” bioburden scenarios is how BIs validate sterilization processes and prove that a process is effective enough to consistently sterilize a product in its final package. Other common sterilization processes validated with BIs are the sterilization of manufacturing equipment and packaging components. BIs are also used to periodically monitor and reassess sterilization processes for their effectiveness.
What is depyrogenation, and why is depyrogenation needed?
Medical devices and parenteral products must be sterile and pyrogen-free. Even if a product is sterile, it can still contain pyrogens. 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. Products can accumulate pyrogens from raw materials or other parts of the manufacturing process. The best pyrogen removal or destruction processes are product-dependent. Standard depyrogenation methods are dry heat, rinsing, and filtration.
What are pyrogens, endotoxins, and lipopolysaccharide (LPS)?
Pyrogens are molecules or substances that cause a feverous reaction when they enter the human body. Endotoxins are the most common type of pyrogen. Endotoxins come from the cell walls of gram-negative bacteria. The endotoxins themselves are molecules with both fat components and complex sugar components. The presence of fat and sugar components is why endotoxins are also known in scientific literature as lipopolysaccharides (LPS). LPS is the biologically active portion of an endotoxin. In other words, LPS is the part of the endotoxin that triggers the innate immune system and causes illness in humans. Parenteral products and devices are contaminated with endotoxin through Gram-negative bacterial cells or cell wall fragments containing LPS. Lipopolysaccharide’s structure allows it to stick to hydrophobic (water-repellant) and hydrophilic (water-loving) surfaces. Thus, LPS components easily attach to molecules and proteins in solutions or material surfaces, causing endotoxin contamination. LPS also sticks to itself to form LPS chains known as aggregates.
What are endotoxin indicators?
Endotoxin indicators (EIs) are endotoxin solutions with a known level of pyrogenic activity. Endotoxin indicators are used to assess depyrogenation process effectiveness by challenging products in a depyrogenation process. Endotoxin indicators can be glass vials inoculated with endotoxin or LPS solutions. All depyrogenation processes, such as rinsing, cleaning, dry heat, and filtration, can be assessed using EIs. Often purified LPS, such as control standard endotoxin lysate (CSE) or reference standard endotoxin (RSE), is purchased from a manufacturer and used for depyrogenation or cleaning validations. However, in-house EIs can be prepared in-house. In-house endotoxin indicators more closely mimic product contamination and provide a realistic assessment of a process’s depyrogenation capability compared to purchased LPS indicators. Indeed, as endotoxin contaminants contain cell membrane and wall components, they aren’t as “sticky” as purified LPS. Thus, endotoxins contaminating parenteral products or medical devices may have less surface adsorption or more stability in solutions or on material surfaces than purified LPS.
What types of biological indicators are there?
Some BIs contain a single microorganism species, while others contain two species and concentrations of bacterial spores to compare different sterilization processes. There are three primary types of BI indicators.
#1: Spores on a carrier
Spores are placed on a carrier (e.g., a disk or strip). The disks or strips can be paper, glass, plastic, metal, or other materials. BI’s using a carrier are often packaged to maintain the integrity and viability of the inoculated spores on the carrier. Following sterilization, neither the BI carrier nor the BI packaging should be damaged or degraded.
#2: Spore suspension
Biological indicators can also be an inoculate spore suspension placed on the product being sterilized or into representative units of the product to be sterilized. A surrogate article that is substantially similar to the product being sterilized can be used if it is not practical to inoculate the actual product. For spore suspensions used in liquid products, the population and D value of the BI spore is critical to determine to evaluate the efficiency of the sterilization process. To inactivate biological spores, a sterilization process must be able to overcome the D value of the BI spore.
#3: Sealed system spores
The third BI option is a sealed system. This sealed system includes the growth medium and the BI microorganisms. The growth medium is either in direct contact with the BI species during the sterilization or is placed in contact with the BI after sterilization. The growth media supports the recovery of any sterilization process-exposed BI microorganisms. Some BI systems contain a growth indicator or sensor in addition to growth media. To inactivate bacterial spores in a sealed system, nearly all spores within the growth media must be destroyed.
What types of endotoxin indicators are there?
Endotoxin indicators require both the endotoxin or LPS standard and an article that carries or houses the standard. The four types of endotoxin indicators used for depyrogenation validations are detailed below.
#1: Endotoxins on a carrier
The best carriers for endotoxins are items that will be depyrogenated, such as glass vials, rubber stoppers, or actual products. Items can be inoculated with endotoxin standard by adding a small volume of a concentrated liquid standard to the carrier or drying the endotoxin onto the carrier. Generally, the concentration of endotoxin or LPS is 1000 EU. However, historical data can justify a higher or lower EU concentration.
#2: Endotoxin suspension
For liquid products, and endotoxin suspension is used to assess the “worst-case” endotoxin contamination (in EU/mL) for the depyrogenation process under evaluation. Worst-case scenarios should take everything into account. Examples of factors to consider are unexpected bioburden from raw materials and longer than normal processing hold times that could cause gram-negative bacteria proliferation.
#3: LPS on a carrier
LPS on a carrier EI is the same as the endotoxins on a carrier EI, except purified LPS is used instead of endotoxins isolated from gram-negative bacteria.
#4: LPS suspension
LPS suspension is the same as endotoxin suspension, except purified LPS is used instead of endotoxins isolated from gram-negative bacteria.
How do you select biological indicators and endotoxin indicators for your sterile product’s processes?
The resistance of the BI to the sterilization process impacts biological indicator selection. The BI system must provide a greater challenge to the sterilization process than the native bioburden of the product being sterilized. The typical BIs are spore-forming bacteria of the genera Geobacillus, Bacillus, and Clostridium. However, other biological indicators may be used with appropriate justification.
EI selection and the subsequent challenge (or inoculation) process should be relevant to the manufacturing process under validation. For example, dry heat depyrogenation frequently uses glass vials or materials with heating characteristics similar to the products under depyrogenation for EI inoculation. However, an endotoxin indicator may not be needed for raw materials with inherently high endotoxin contamination to validate depyrogenation. An endotoxin indicator may not be necessary because the original contamination level is sufficient to compare against downstream depyrogenated materials accurately. As another example, depyrogenation of raw materials or upstream intermediates without high endotoxin contamination using highly purified LPS from RSE or CSE might not reflect the actual reduction potential of product depyrogenation due to LPS purity. Thus, endotoxins harvested from gram-negative bacteria would represent the gram-negative cell wall fragments (and outer membrane constituents) depyrogenation filters would actually need to process.
Either a recognized gram-negative bacterial strain or an isolated organism may be used to create endotoxin standards. Gram-negative organisms may be isolated from a facility, raw material, water source, or product. Isolated organisms will require species-level identification, and the endotoxins’ activity will need to be determined. RSE or CSE can be used for LPS standards and have known endotoxin activity measured in endotoxin units (EU).
Summary
Overall, sterilization is any process that removes, kills, or deactivates all forms of life, while depyrogenation is a process that eliminates pyrogens. Most medical devices, parenteral products, and other health care items must be sterile and pyrogen-free to be used by a patient. Sterilization processes for healthcare products are validated using biological indicators (BIs). In contrast, endotoxin indicators (EIs) are used to validate depyrogenation processes and assess pyrogen removal effectiveness. A biological indicator is a preparation of one or more spore-forming microorganisms that have resistance to being killed during sterilization. Endotoxin indicators are endotoxin or highly pure LPS solutions with a known level of pyrogenic activity. To inactivate biological spores, a sterilization process must be robust enough to destroy most of the BI spores.
Typical BIs are gram-positive bacteria of the genera Geobacillus, Bacillus, and Clostridium. When selecting a BI for sterilization validation, the BI strain should have greater resistance to the sterilization process than the product’s native bioburden. There are no recommended gram-negative bacterial strains for endotoxin indicators, as each product will be exposed to different gram-negative species and endotoxin contaminants. EI selection and the subsequent challenge (or inoculation) process should be relevant to the manufacturing process under validation. For raw materials or intermediates with inherently high endotoxin contamination, endotoxin indicators may not be required to validate depyrogenation. All in all, ensure you choose a contract testing organization that can provide appropriate sterilization validations, sterility testing, and bacterial endotoxin testing for your product needs.
Ethide Labs is a contract testing organization specializing in Sterilization Validations. Ethide Labs also offers Microbiology Testing, Bioburden Testing, Bacterial Endotoxin Testing, EO Residual Testing, Sterility 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.
International Organization for Standardization. Sterilization of health care products- Moist heat- Part 1: Requirements for the development, validation, and routine control of a sterilization process for medical devices. Geneva (Switzerland): ISO; 2006. (ISO 17665-1:2006/(R)2016).
Michael J. Akers. Sterile Drug Products Formulation, Packaging, Manufacture, and Quality. Drugs and the Pharmaceutical Sciences. Informa Healthcare. 2010.
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United States Pharmacopeial Convention. <1115> Bioburden Control of Non-Sterile Drug Substances and Products. Rockville, MD, USA. 2021. (USPC <1115>).
United States Pharmacopeial Convention. <1116> Microbiological Control & Monitoring of Aseptic Processing Environments. Rockville, MD, USA. 2021. (USPC <1116>).
United States Pharmacopeial Convention. <1211> Sterility Assurance. Rockville, MD, USA. 2021. (USPC <1211>).
United States Pharmacopeial Convention. <1228.5> Endotoxin Indicators For Depyrogenation. Rockville, MD, USA. 2021. (USPC <1228.5>).
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