Microbial Aerosol Challenge Testing vs. Bioburden 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 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 in a given area or 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 effort of the human immune system.
What is bioburden testing?
Bioburden testing measures the viable organisms present in a sample. The total viable organisms present is known as a total viable count. This total viable count (expressed as CFUs) provides a value for the microbial contamination level within a particular sample. Bioburden is used to identify the number of live microorganisms present in a sample from a product, package, or manufacturing environment. Bioburden testing follows the methods outlined in USP 60, USP 61, and USP 62.
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 bioburden testing?
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 sources of contamination, regular bioburden testing of all sterile products reduces microbial contamination and recall risks.
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 are bioburden tests performed?
Microbiology and bioburden testing methods overlap. Both microbiology and bioburden tests require sample collection from a product, a surface, a water source, or the air. Once samples are obtained, the microbial content (bioburden) can be quantified. Below are links to articles related to multiple microbiology tests and testing methods. Bioburden microbial characterization methods can be found here, whereas the top techniques for microbiology examination are here. A comparison between USP 60, USP 61, and USP 62 bioburden testing regulations can be found here.
What are the differences between microbial aerosol challenge testing and bioburden testing?
Microbial aerosol challenge testing tests the package integrity of filled vials. In contrast, bioburden testing quantifies the microbes within an environment or a product. Microbial counting methods for both tests are similar. However, microbial aerosol challenges use a single bacteria type (Bacillus atrophaeus) to evaluate the integrity of vial elastomeric stoppers for multidose products. In contrast, bioburden testing, like microbiology testing, can evaluate multiple microbes. For distinctions between bioburden and microbiology testing, please visit our article here.
Summary
Overall, microbial aerosol challenge testing and bioburden testing are imperative for regulatory approval of parenteral products, medical devices, and combination products. Microbial aerosol challenge testing ensures that multidose medical devices, products, and therapies have sufficient packaging integrity and to keep patients safe during product use. In contrast, bioburden testing evaluates the microbial levels of products and manufacturing environments at various phases during production and packaging. Bioburden content supports monitoring and controlling microbes for sterilization and aseptic processes. All in all, ensure you choose a contract manufacturing organization that can support you with appropriate microbial aerosol challenge testing and bioburden testing for your unique parenteral product, medical device, or combination product needs.
Ethide Labs is a contract testing organization that specializes in Bioburden Testing and has access to Microbial Aerosol Challenge Testing. Ethide Labs also offers Microbiology Testing, Sterility Testing, Bacterial Endotoxin Testing, Ethylene Oxide 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
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).
Michael J. Akers. Sterile Drug Products Formulation, Packaging, Manufacture, and Quality. Drugs and the Pharmaceutical Sciences. Informa Healthcare. 2010.
Technical Report No. 27, Pharmaceutical Packaging integrity, 1998, PDA.
Technical Information Bulletin No. 4, Aspects of Container/closure Integrity, PDA.
United States Pharmacopeial Convention. <60> Microbiological Examination of Nonsterile Products- Tests for Burkholderia Cepacia Complex. Rockville, MD, USA. 2021. (USPC <60>).
United States Pharmacopeial Convention. <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests. Rockville, MD, USA. 2021. (USPC <61>).
United States Pharmacopeial Convention. <62> Microbiological Examination Of Nonsterile Products: Tests For Specified Microorganisms. Rockville, MD, USA. 2021. (USP <62>).
