Rapid Sterility Testing

Your SteriSEQ assay order includes:

• SteriSEQ Assay Mix, 2 x 110 μL: Store at -25°C to -15°C and protect from light
• qPCR Master Mix Plus, 2 x 750 μL: Store at -25°C to -15°C until first thawed, then store at 2°C to 8°C and protect from light
• SteriSEQ Discriminatory Positive Control, 2 x 30 μL: Store at -25°C to -15°C
• SteriSEQ DNA Dilution Buffer, 1 x 7 mL: Store at -25°C to -15°C until first thawed, then store at room temperature

The SteriSEQ assay is a real-time, quantitative PCR (qPCR) based assay for sensitive and specific detection of bacterial and/or fungal DNA in test samples. The assay utilizes TaqMan qPCR assays that target conserved regions in a broad panel of either bacterial or fungal genomes. Although the SteriSEQ assay is intended for use as a presence/absence test, because it utilizes qPCR, changes in target DNA levels in test samples can be monitored over time. This feature enables assessment of viability of organisms in positive test samples, where increasing levels of target DNA, as measured by drop in Ct, indicate replication of the detected microorganisms. 

The SteriSEQ Rapid Sterility assay inclusion panel includes over 16,000 bacterial species and 2,600 fungal species.  For bacteria, the panel includes gram-positive, gram-negative species, anaerobic, aerobic species. A full list of the inclusion panel is available. 

The SteriSEQ assay is not an identification test. The assay uses 2 primer probe sets, one for bacteria and one for fungus, so the assay discriminates at that level. There can be scenarios where extracted DNA that tests positive by the SteriSEQ assay can be further analyzed with MicroSEQ ID to provide an identification to the species level. An example would be a sample that tests positive with the SteriSEQ assay, the contamination is only from one species (or only one bacterial and one fungal species) and contains a level of DNA (as assessed by Ct value) that enables amplification and sequencing in the MicroSEQ ID workflow. 

Sterility testing of biological drug products is a regulatory requirement. It helps to ensure patient safety by testing for any harmful microorganisms and fungus, that may contaminate a drug product. Early detection of contamination allows for timely intervention, such as discarding contaminated batches or implementing corrective actions.

The U.S. Food and Drug Administration (FDA) defines "sterile" as the absence of all viable microorganisms in the sample tested. This typically means that a product or substance has undergone a sterilization process to ensure it is free from any living bacteria, viruses, fungi, or other microorganisms. And that the product has been tested with an accepted sterility test method and passed that test. This definition is critical for products such as pharmaceuticals where sterility is essential to ensure safety and efficacy. The FDA provides guidelines and regulations on methods and validation processes both to achieve and maintain sterility in such products and also on tests for sterility. 

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 providing the test is negative at the time of infusion.

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

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.

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.

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 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 testing for microbial contamination (e.g., bacteria, yeast, mold) should be conducted at multiple stages.

The SteriSEQ system was built as a four-plex assay. We also needed a passive reference dye for the 7500 Fast system to display the amplification plots. Mastermix option was limited to bactopure (ROX passive reference) due to the sensitive nature of the product. FAM and VIC 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.