Important News:  Ethide is now a part of Millstone Medical Outsourcing. Learn more

Microbial Aerosol Challenge Testing vs. Microbiology 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 microbiology?

Microbiology contains the Greek and Latin roots of “micro,” “bio,” and “ology.” The “micro” stands for small, the “bio” stands for biology, and the “ology” is the study of something. Microbiology can also be defined by “microbial,” which refers to microorganisms. Thus, microbiology is the study of microorganisms, where microorganisms are small living components of the broad field of biology.

What is microbiology testing?

Microbiology testing identifies the presence and the type of microbes in a manufacturing environment, medical product, or medical device. Specific regulatory testing for microbiology may include assays such as microbial enumeration testing, particulate analysis, yeast analysis, antifungal activity assessment, growth promotion testing, microbial limits testing, zone of inhibition testing, water analysis, bacteriostasis/fungistasis testing, bacteria identification, yeast identification, fungi identification, gram-negative staining, product inoculations, biological indicator (BI) tests, and BI incubation time reduction studies. Microbiology tests are governed by USP 61 and USP 62.

Why Are Microbiology Testing And Microbial Aerosol Testing Important?

Microbiological testing is imperative for preventing product-induced infections and mitigating contamination risk in manufacturing environments, especially aseptic manufacturing environments. Aseptic manufacturing environments rely heavily on microbiological testing because aseptic processing requires excluding microorganisms from the manufacturing methods. Thus, microorganisms must be prevented from entering open containers or product materials during aseptic processing. As a result, microbial tests monitor clean rooms and facilities that manufacture products using aseptic processing.

Microbial aerosol challenge tests evaluate if external bacterial contaminants can enter a medical product’s sterile packaging system. Microbial aerosol challenge testing is essential for verifying multi-use vial enclosure systems (such as those used for vaccines and other parenteral products).  All microbial aerosol challenge methods are performed in an ISO Class 5 laminar flow hood to maintain sterility during the assessment. 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. After aerosol challenge testing, vials with robust microbial barriers will not have bacterial growth within the vial.

Picture of a microscope, several flasks and test tubes, and other laboratory stuff on a laboratory table with a blue background. Microbiology testing for parenteral products and combination products. Microbial challenge for parenteral products and combination products. Microbiology tests regulations. Manufacturing methods for parenteral products and combination products. Microbiology tests and parenteral products, and combination products

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 microbiology testing?

Microbiological testing is imperative for preventing product-induced illness and mitigating contamination risk in manufacturing environments, especially aseptic manufacturing environments. Aseptic manufacturing environments rely heavily on microbiological testing because aseptic processing requires the exclusion of microorganisms from the manufacturing methods. Thus, microorganisms must be prevented from entering open containers or product materials during aseptic processing. As a result, microbial tests help monitor clean rooms and facilities that manufacture products using aseptic processing. All sterile products and medical non-sterile products will require one or more microbial tests before marketing.

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 microbiology tests performed?

Microbiology testing often requires sample collection from a product, a surface, a water source, or the air. Once samples are obtained, various microbiology testing can be performed. Below are links to articles related to multiple microbiology tests and testing methods. Firstly, further details on USP 788 particulate matter testing can be found here. Microbial characterization methods can be found here, whereas the top techniques for microbiology examination are here. Lastly, information on how to record and evaluate microbiology data is here.

What are the differences between microbial aerosol challenge testing and microbiology testing?

Microbial aerosol challenge testing tests the package integrity of filled vials. In contrast, microbiology testing is a test for environmental monitoring and sterility assurance. 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, microbiology testing evaluates multiple microbes.

Summary

Overall, both microbial aerosol challenge testing and microbiology 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 keep patients safe during product use. In contrast, microbiology testing provides sterility assurance of the product’s manufacturing environment and the product itself. 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.

Ethide Labs is a contract testing organization that specializes in Microbiology Testing and has access to Microbial Aerosol Challenge Testing. Ethide Labs also offers Bioburden Testing, Sterility Testing, Bacterial Endotoxin Testing, Ethylene Oxide Residual Testing, Cytotoxicity Testing, Environmental MonitoringPackage 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).

Technical Report No. 27, Pharmaceutical Packaging integrity, 1998, PDA.

Technical Information Bulletin No. 4, Aspects of Container/closure Integrity, PDA.

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>).

Share this in your social networks