Guideline To Create Your Own Endotoxin Indicator For Regulatory Testing
What Are Endotoxin Indicators And 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. A patient exposed to endotoxins from a medical device or injection can go into endotoxin shock. Thus, medical products undergo a bacterial endotoxin test to ensure no endotoxins are present before product use. If a bacterial endotoxin test indicates that endotoxins are present, endotoxin removal can be completed through depyrogenation process that will either destroy or remove the bacterial endotoxins. Endotoxin indicators are used during endotoxin removal and bacterial endotoxin tests to evaluate the amount of endotoxin present in a medical product.
Why is endotoxin removal needed And What is endotoxin shock?
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. If the human body is exposed to pyrogens, patients can go into endotoxin shock. Endotoxin shock is a form of septic shock, where common symptoms are fever and chills. Endotoxin shock can be deadly. To avoid endotoxin shock, endotoxin removal processes are used on medical devices and sterile products. The best endotoxin removal or destruction process (known as depyrogenation) depends on the product. Standard depyrogenation methods are dry heat, rinsing, and filtration. A comparison between endotoxin removal via rinsing versus dry heat can be found HERE. Comparisons between endotoxin removal through filtration versus dry heat or rinsing can be found HERE and HERE, respectively.
What are pyrogens?
Pyrogens are molecules or substances that cause a feverous reaction when they enter the human body. Endotoxins are the most common type of pyrogen. Regulatory tests for pyrogens can be found HERE.
What is an endotoxin?
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). Endotoxins can be deadly to humans because they trigger the innate immune system, causing illness.
What is lipopolysaccharide (LPS)?
LPS is the biologically active portion of an endotoxin. LPS is amphipathic and has three distinct regions. The first is a water-repellant (hydrophobic) lipid-A portion that provides lipopolysaccharide’s biological activity. The lipid-A component is essentially the same among Gram-negative bacteria species. The second region is a water-loving (hydrophilic) O-antigen, which varies based on Gram-negative bacteria strain. The final part is an oligosaccharide linkage between the lipid-A and O-antigen potions of LPS.
How do endotoxins and lipopolysaccharides (LPS) contaminate medical devices and parenteral products?
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 (nonpolar) and hydrophilic (polar) 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. Temperature, pH, salt concentration, and other factors affect the biological activity and stability of the solution’s endotoxins (or LPS).
What are endotoxin indicators?
Endotoxin indicators (EIs) are endotoxin solutions with a known level of pyrogenic activity. EIs are used to assess depyrogenation process effectiveness by challenging products in a depyrogenation process. EIs 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 aren’t as “sticky” as purified LPS, endotoxins contaminating parenteral products or medical devices may have less surface adsorption or more stability in solutions than purified LPS.
How do you select, prepare, and use endotoxin indicators for process validation?
Endotoxin Indicator Selection
The 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.
Endotoxin Indicator Preparation
Fillers or excipients should not be included in endotoxin indicator preparations. Purchased CSE and RSE have set endotoxin unit (EU) activity and do not require additional preparation before use. However, preparing an endotoxin standard in the laboratory requires additional effort. Either a recognized gram-negative bacterial strain or an isolated organism may be used. 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. The relative activity of the endotoxin preparation can be determined by comparing its activity to known RSE or CSE LPS standards. Once the relative activity is confirmed, the endotoxin must be tested with a bacterial endotoxin test to ensure the endotoxin reacts with the limulus amoebocyte lysate (LAL) as LPS does.
The activity of the stock endotoxin preparation is reported as:
S = (Test result in EU/mL) × (DF)
Where:
DF = dilution factor
S = starting endotoxin preparation activity (EU/mL)
For isolated organisms, the following items will need to be addressed:
- The proper maintenance of the organism
- The growth conditions, including any requirements to prepare media, nutrients, incubation temperature, and incubation time
- Cryopreservation or lyophilization methods
- Endotoxin storage methods including concentration (in EU/mL), storage volume, storage vessel type, the temperature of storage, and the length of storage
- Data on the stability of the preparation (which will determine endotoxin expiry)
- Batch production records should be written, managed via change control, and followed.
Endotoxin Indicator Inoculation & Use
Endotoxin indicators require both the endotoxin or LPS standard and an article that carries or houses the standard. The best carriers 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 be used to justify a higher or lower EU concentration. For liquid endotoxin carriers, the level of inoculation in EU/mL should be justified based on “worst-case” endotoxin contamination for the depyrogenation process under evaluation. Worst-case scenarios should take everything into account, including unexpected gram-negative bioburden from raw materials and longer than normal processing hold times that could cause gram-negative bacteria proliferation.
How do you determine depyrogenation effectiveness?
Depyrogenation process effectiveness is determined by comparing the endotoxin activity after depyrogenation to the endotoxin or LPS activity of unprocessed EI controls. In the comparison, the log10 of the endotoxin concentration after depyrogenation is subtracted from the log10 of the original EI concentration. The subtraction result is the log reduction of the depyrogenation process. At least a 3-log reduction is required by most regulatory bodies for valid depyrogenation processes. However, the log reduction requirements for depyrogenation are process- and product-dependent.
Summary
Depyrogenation is a process that removes pyrogens. Testing for pyrogens is an imperative safety metric for regulatory approval of medical devices and parenteral products. The most prevalent and problematic pyrogens are the bacterial endotoxins (also known as LPS) found in the outer cell walls of gram-negative bacteria. Endotoxin indicators (EIs) are endotoxin solutions with a known level of pyrogenic activity. EIs are used to assess depyrogenation process effectiveness. 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, an endotoxin indicator may not be required to validate 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. <1228.5> Endotoxin Indicators For Depyrogenation. Rockville, MD, USA. 2021. (USPC <1228.5>).
