Towards Rapid Microbial Testing Methods

Given the highly specialized nature of CAR-T treatments and the immunocompromised state of the patients receiving them, ensuring the sterility of these therapies is of utmost importance. 

Sterility testing is particularly difficult for autologous cell and gene therapies because of limited volumes and the need for speed; ensuring timely delivery for patient treatment is a priority. More rapid sterility testing also has an advantage in frozen CAR-T products because it accelerates the time from products to patients. 

The compendial sterility test, which involves direct inoculation of tested product into the medium (1), is recognized by the industry for its adherence to regulatory standards and for its reliability; however, it takes 14 days.

To shorten time, we have developed and qualified a rapid sterility test using the BD Bactec FX 40 device for early detection of potential contamination in frozen CAR-T drug products (2). In this study, we assessed the comparability of our method with the compendial sterility test and validated the method by sensitivity and specificity assessment in accordance with the regulatory requirements. In short, it offers an automated and rapid microbiological solution that aligns with the adoption of efficient microbiological methods for advanced therapies (3). Below, we present a summary of our work. 

First, we optimized the culture conditions, including the addition of fastidious organism supplement (FOS), and the technique of vial inoculation, particularly for anaerobic strains (for example, Clostridium sporogenes), which are sensitive to manipulation.

For both the comparability study and the validation study, we used nine microbial reference strains (aerobic and anaerobic bacteria, and fungi, namely Clostridium sporogenes, Cutibacterium acnes, Staphylococcus aureus, Streptococcus pyogenes, Brachybacterium nesterenkovii, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus brasiliensis) as listed in Ph.Eur  Chapter 2.6.1 and USP <71>. Three in-house isolates (aerobic bacteria) were also included in the testing.

The microbial reference strains were inoculated to the BD Bactec FX40 vials in concentrations of 100, 50, and 10 colony-forming unit (CFU)/vial, together with the FOS. Concentration 100 CFU was tested on three batches of frozen CAR-T drug product, whilst concentrations of 50 CFU and 10 CFU were tested in triplicates of each microorganism on one batch of frozen CAR-T drug product. All vials were cultivated in the BD Bactec FX40 at temperature 35 ± 1,5°C up to 14 days or until detection of microbial growth (positive status in the device). 

For the comparability study, results in 100 CFU inoculated samples were compared between the rapid sterility method and the compendial method. Both methods were tested in parallel, and all tested microbial strains showed positive results within four days – a time frame comparable with the compendial method. All types of negative control were without growth after 14 days. Three in-house isolates from the clean room environment were also tested with positive results.

For the method validation, the sensitivity and the specificity comparison between the two methods was performed using bacterial and fungal contaminants at 10 CFU and 50 CFU/vial concentrations. Specificity was verified through samples spiked with 50 CFU of micro-organisms (positive) and non-spiked samples (negative).

Our study confirmed that the rapid sterility test effectively detected bacteria or fungi inoculated at 10 CFU, while the compendial sterility test did not always yield positive results at this concentration. The time to positivity for all microbial strains was within four days, indicating the rapidity and efficiency of the Bactec method. The method’s specificity was also confirmed, as it successfully detected negative samples. Moreover, all microorganisms detected by the BD Bactec FX40 device were successfully grown in subsequent subcultures, validating the reliability of the method. 

Nevertheless, we encourage the testing of other cleanroom-specific environmental bacterial or fungal species. Strains from the environment that require lower culture temperature can be challenging to grow, but the issue can be overcome by parallel testing with agar plates (4). To ensure maximum safety, a conservative approach of combining Bactec testing with the compendial method is recommended for conditional product release after five days of Bactec testing, followed by the compendial sterility test (after 14 days) for the final release.

In 2020, NIST launched the Rapid Microbial Testing Methods (RMTM) Consortium (5). Input from the presenters, panelists, and attendees emphasized the need for the community to work together to efficiently overcome challenges that limit the use of RMTMs. In the meantime, patients remain in critical need, so industry must evaluate and promote methods that ensure quick deployment of safe therapies.