Microbial Aerosol Challenge Testing vs. Bacterial Endotoxin Testing
What is microbial aerosol challenge testing?
Microbial aerosol challenge testing evaluates the enclosures of filled product vials to ensure that the vial enclosure does not allow microbes to enter following the entry and exit of syringe needles during dosage draws. In a microbial challenge, a product, agent, or process is exposed to (challenged by) microbes.
What is a bacterial endotoxin?
Endotoxins come from the cell walls of gram-negative bacteria. The endotoxins themselves are molecules with fat components and complex sugar components (also known as polysaccharides). Therefore, endotoxins are also known as lipopolysaccharides (LPS) in scientific literature. Endotoxins are also considered pyrogens because they trigger the innate immune system and produce fever when released within the human body.
What is bacterial endotoxin testing?
A bacterial endotoxins test (BET) uses an assay known as the Limulus Amoebocyte Lysate (LAL) test. BET testing is considered a pyrogenicity test. However, do not confuse BET testing with rabbit pyrogen 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.
What products require microbial aerosol challenge testing?
Microbial aerosol challenge testing is essential for verifying multi-use vial enclosure systems (such as those used for vaccines and other parenteral products). Microbial aerosol challenge testing ensures that the sterile or aseptic vials act as an effective microbial barrier and preserve the product’s sterility even when the outside is exposed to high concentrations of bacteria.
What products require bacterial endotoxin testing?
Cosmetics do not require bacterial endotoxin testing, but FDA-regulated medical products (such as medical devices, parenteral products, and combination products) require BETs. Bacterial endotoxin testing is an important quality control step that detects product endotoxin contamination levels at any production stage, from initial product manufacture to final distribution. As 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.
How is microbial aerosol challenge testing performed?
Microbial aerosol challenge testing evaluates a finished product’s final sterile barrier system’s package integrity by exposing the terminally sterilized, packaged product to aerosolized Bacillus atrophaeus bacteria. After the package exterior has been exposed to Bacillus atrophaeus, the package contents are tested for bacterial contamination.
Microbial aerosol challenge testing begins with the preparation of Trypticase Soy Broth (TSB) media that will be used as a negative control (without bacteria) and positive control (with bacteria). All microbial aerosol challenge methods are performed in an ISO Class 5 laminar flow hood to maintain sterility during the assessment. All positive controls, negative controls, and samples will have adapters for syringe use. For the preconditioning process, ten samples (terminally sterilized vials containing product) and all controls are held statically in an ISO Class 7 controlled environment for 28 days. Five samples are laid down on their side, and five are left standing on their base during the 28 days of storage. Two dose removals will be performed from the TSB vial at 0, 7, 14, 21, and 28 days. After 28 days of sample conditioning, the vial adapter/syringe assembly will undergo the microbial aerosol challenge test.
A Bacillus atrophaeus suspension is prepared for the microbial challenge so that the aerosol chamber receives a minimum of 1.0 x 107 spores when the bacterial is aerosolized. The final suspension concentration is verified using the spread plate method. Once the aerosol chamber with Bacillus atrophaeus suspension has been prepared, the ten test media sample vials with attached syringe-adapter assemblies are loaded, equally spaced and upright, into the aerosol chamber along with the positive controls. Positive controls are created by piercing the vial septum with a sterile 23-gauge needle and leaving the needle in place during the microbial challenge. Negative control vials are not loaded into the chamber. Once all positive controls and sample vials are loaded into the aerosol chamber, the doors are sealed, and the chamber fan is turned on. The prepared Bacillus atrophaeus suspension is loaded to the chamber’s nebulizers, and the suspension is aerosolized until the nebulizers are empty. Samples are left in the chamber for an hour after the suspension has been fully aerosolized. Next, samples are taken out of the chamber, and the exterior is thoroughly disinfected. Finally, all positive control, negative control, and test vials are incubated upright for seven days.
After seven days, all control and sample vials are checked for microbial growth. The level of microbial growth is assessed by streaking the samples onto TSA agar and incubating them. Any growth on the agar plates is compared to Bacillus atrophaeus by Gram staining and direct microscopic observation. Negative TSB vial sterility results indicate that the sterile barrier vial system acted as an effective microbial barrier for the product under the microbial aerosol challenge testing conditions. Positive TSB media vial results indicate that the sample closures were not an effective microbial barrier.
How is bacterial endotoxin testing performed?
LAL is dissolved in a water or buffer solution free of endotoxins and interfering factors to form a standard endotoxin stock solution. All endotoxin stock solutions are calibrated to the current World Health Organization International Standard for Endotoxin. Endotoxin is expressed in Endotoxin Units (EU). One USP Endotoxin Unit (EU) is equal to one International Unit of endotoxin. Then a serial dilution of the endotoxin standard is prepared. Solutions for samples to be tested are prepared by immersing the device or dissolving the product in the same water or buffer solution used for the endotoxin stock solution. If necessary, the pH of the solution will be adjusted so that it falls within the range of 6.0-8.0.
A LAL gel-clot technique is used for detecting or quantifying endotoxins based on lysate reagent clotting upon endotoxin exposure. A series of tests are performed to confirm the sensitivity of the lysate solution and for any interfering factors. The sensitivity of the lysate solution is considered to be the minimum concentration of endotoxin required to cause the lysate to clot under test conditions. Both quantitative and limit testing using the LAL gel-clot technique can be performed. For brevity, only the bacterial limit test is described below.
For bacterial endotoxin limit testing of your medical device, four solutions (Solutions A, B, C, and D) are prepared. The details of these solutions are shown in Table 1 of USP 85. Solutions B and C are positive controls with standard endotoxin solution at twice the concentration of the lysate sensitivity. Solution D is a negative control that consists of water or buffer solution.
A BET test is valid when all Solutions B and C samples test positive and all Solution D samples are negative. In other words, the test is accurate when the positive controls test positive and the negative controls test negative. If both Solution A samples test negative for bacterial endotoxin, the samples pass the BET test limits. If both Solution A samples test positive for endotoxin, the samples fail the BET test. The BET test is repeated if one of the Solution A samples tests positive and the other tests negative. Medical device samples do not comply with the BET testing limits if a positive test result is found for one or both Solution A sample replicates.
Amoebocyte lysate reacts to endotoxins and some β-glucans. Amoebocyte Lysates that do not respond to glucans are available. These amoebocyte lysates have the G factor reacting to glucans removed or inhibited. If you are concerned about the presence of glucans in your samples, ensure that an amoebocyte lysate is used. LAL testing has special considerations for product families and special medical device endotoxin limits. There are also endotoxin limits for drugs and biologics. Should your products be above their specified limits, endotoxin controls and depyrogenation studies are available to support reducing a product’s endotoxin concentration. In rare cases, traditional LAL testing may not be possible, and in-vivo pyrogen testing governed by USP 151 may be needed.
What are the differences between microbial aerosol challenge testing and bacterial endotoxin testing?
Microbial aerosol challenges and endotoxin tests are very different. Microbial aerosol challenges use a single bacteria type (Bacillus atrophaeus) to evaluate package integrity for multidose products. In contrast, bacterial endotoxin testing evaluates product contents for endotoxins. Overall, microbial aerosol challenge testing is a package integrity test for products filled in vials, while bacterial endotoxin testing is a product safety test.
Summary
Overall, microbial aerosol challenge testing and bacterial endotoxin testing are imperative for regulatory approval of parenteral products, medical devices, and combination products. Microbial aerosol challenge testing ensures that multidose medical devices, drug or biologic combination products, and therapies have sufficient package integrity and keep patients safe from microbial contamination during product use. In contrast, bacterial endotoxin testing evaluates product contents for endotoxins. All in all, ensure you choose a contract manufacturing organization that can support you with appropriate microbial aerosol challenge testing and microbiology testing for your unique parenteral product, medical device, or combination product needs.
MycoScience is a contract manufacturing organization specializing in sterile syringe and vial filling cosmetic products or for products used in animal studies. In addition, MycoScience offers testing services, including Preservative Efficacy Testing, Cytotoxicity Testing, Bioburden Testing, Cleaning Validations, Microbial Aerosol Challenge Testing, Accelerated Aging, Microbiology Testing, EO Residual Testing, Bacterial Endotoxin Testing, Package Integrity Testing, Sterilization Validations & Environmental Monitoring services medical devices and allied industries. MycoScience is an ISO 13485 certified facility.
References
ANSI/AAMI/ISO 11607-1, 2006/A1:2014, Packaging for terminally sterilized medical devices – Part 1: Requirements for materials, sterile barrier systems, and packaging, Amendment 1.
ASTM F1608 – 16, Standard Test Method for Microbial Ranking of Porous Packaging Materials (Exposure Chamber Method).
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.
Technical Report No. 27, Pharmaceutical Packaging integrity, 1998, PDA.
Technical Information Bulletin No. 4, Aspects of Container/closure Integrity, PDA.
United States Pharmacopeial Convention. <85> Bacterial Endotoxins Test. Rockville, MD, USA. 2021. (USPC <85>).
