SteriSEQ™ Rapid Sterility Testing Kit

The SteriSEQ Rapid Sterility Testing System was built as a four-plex assay. We also needed a passive reference dye for the 7500 Fast Real-Time PCR System to display the amplification plots. Mastermix option was limited to bactopure (ROX passive reference dye) due to the sensitive nature of the product. FAM and VIC dyes were for fungi and bacteria targets, which were both MGB probes. For multiplex assays, the recommendation is not to use more than 2 MGB probes, so we were limited to QSY dyes for IPC and DPC. It is known that ABY channel tends to cross to ROX and having DPC in ABY channel caused ROX signal to be pulled up for high copy DPC. Therefore, IPC was placed in ABY channel since IPC levels are consistent across wells. This leaves DPC in Alexa/Cy5 channel. Alexa gave a stronger signal and therefore was chosen for DPC.

The SteriSEQ Rapid Sterility test excels in several in-process testing points, for example:

Cell isolation and initial culture setup:
Sterility of source materials: At the beginning of the process, cell sources are collected. These source materials may be a potential point of contamination and regulatory bodies recommend that they are tested for microbial contaminants before being processed. Contaminants could be introduced from leukapheresis starting material or during handling.

Cell expansion and culture:
Since cells are cultured and expanded over time, in-process sterility testing for microbial contamination (e.g., bacteria, yeast, mold) should be conducted at multiple stages.

In-process sterility testing enables the production process remains under control and microbial contamination risks are minimized before final product release. Testing helps ensure the overall safety and quality of the cell therapy product. Detecting contamination early in the manufacturing process (e.g., during cell expansion) can help prevent the continuation of a compromised batch, saving time and resources. This reduces the risk of wasting materials, labor, and other costs associated with manufacturing and testing.

Regulatory agencies, including the FDA, accept qPCR and nucleic acid-based testing (NAT) for sterility as long as they are appropriately validated and produce comparable results to the compendial method within allowable limits established on a case-by-case basis . The FDA's 2024 guideline on ''Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products'' specifies that sterility testing should comply with USP Chapter <71> or use an alternative test method validated according to USP <1223>. USP <1223> provides further guidance on validating alternative methods, such as nucleic acid-based tests for sterility testing. Additionally, USP <1071> and Ph. Eur. 2.6.27 outline a risk-based approach for selecting rapid microbial testing methods, including NAT, for products with short shelf lives.

Cell-based and other living therapies are unique as they cannot be terminally sterilized using heat, filtration, or other methods. Many of these therapies have short shelf lives and often the patient requires infusion as soon as possible following final formulation due to their disease state. Because of this, the FDA and other regulatory agencies can make exceptions to typical sterility requirements. For example, the FDA allows infusion of patients prior to the completion of rapid or traditional growth-based sterility tests provided the test is negative at the time of infusion.

Regarding growth-based sterility testing, the reason growth-based tests take 7 to 14 days or longer is because it takes that long to detect the slowest growing organisms. Fast growers such as E. coli can be detected sooner. A negative result is based on how long it takes to detect the most challenging species.