Rapid sterility testing for advanced therapy manufacturing 

1 Rapid sterility testing to keep pace with cell and gene therapy manufacturing As advanced therapies become more personalized and time-sensitive, sterility testing is evolving to support rapid product release without compromising patient safety. I n cell and gene therapy manufacturing, time is tightly tied to product viability, patient schedules, and operational risk. This is especially evident in sterility testing, a step that is essential for patient safety but increasingly difficult to align with the pace of advanced therapy medicinal products (ATMPs). For decades, compendial sterility tests described in USP <71> have served as the industry standard. These culture-based methods are well established and widely accepted, but they typically require up to 14 days to confirm product sterility. For many ATMPs — often personalized, short-lived, and produced under tight timelines — this timeline is no longer practical. As the ATMP pipeline continues to expand, manufacturers are seeking sterility testing solutions that maintain confidence and compliance while matching the speed of modern production. Unique microbial detection challenges in ATMPs Sterility testing for ATMPs is complicated by the nature of the products themselves. Many ATMP formulations contain high concentrations of living cells. Cellular debris, extracellular matrix components, and formulation excipients can interfere with molecular assays, reducing sensitivity or generating ambiguous signals. At the same time, manufacturers are typically limited to testing small sample volumes, leaving little opportunity for repeat analysis if results are inconclusive. Compounding the issue, not all microbial contaminants behave predictably under culture-based conditions. Some clinically relevant organisms grow very slowly, while others may not grow at all in standard sterility media. DNA-based molecular detection methods like PCR can help overcome these limitations, but they introduce new challenges: bacterial and fungal DNA is ubiquitous in laboratory environments and reagents, making it difficult to distinguish true contamination from background noise. For quality control teams, ambiguous results carry tangible consequences. A false negative can compromise patient safety, while a false positive can delay product release, disrupt manufacturing schedules, and trigger extensive investigations. As a result, there is a growing need for methods that not only deliver results more quickly but also provide greater accuracy. Bringing speed and precision to sterility testing Digital PCR (dPCR) offers a fundamentally different approach to microbial detection. Rather than amplifying targets within a single bulk reaction, dPCR partitions each sample into thousands of individual reactions. Each partition either contains a target molecule or does not, allowing absolute quantification based on statistical analysis. This partitioning enables true microbial signals to stand out clearly from low-level background contamination, even in complex samples. Building on this principle, Sartorius developed the Cyclus® dPCR Tool Box Bacteria Fungi, a sterility detection solution designed specifically for time-sensitive ATMP applications. The assay targets highly conserved regions of bacterial 16S rRNA and fungal 18S rRNA genes, enabling broad organism coverage while maintaining specificity. In silico analysis 2 shows coverage of at least 94% of sequenced bacterial species, including all microorganisms relevant to USP <71> sterility testing standards. Critically, the Cyclus® assay enables sterility results to be generated within a few hours, supporting faster release decisions for time-sensitive products. Performance studies have demonstrated reliable detection down to 10 colony-forming units (CFU) per milliliter, even in challenging matrices such as CAR T products containing approximately 10 million cells per milliliter. Implementing rapid sterility testing in the QC laboratory Beyond analytical considerations, rapid sterility testing methods must also fit smoothly into existing quality control environments. The Cyclus® dPCR Tool Box is designed to work with widely used digital PCR platforms from multiple vendors, including both droplet- and nanoplate-based systems, allowing laboratories with existing dPCR capabilities to adopt the assay without major capital investment. To support consistent and reliable results, the workflow pairs the Cyclus® assay with the Microsart® ATMP Extraction Kit, which is optimized for efficient DNA extraction from cell-based products. The kit format includes lyophilized, highly pure reagents and color-coded tubes, simplifying handling and reducing the risk of operator error. Internal controls built into the assay help identify issues related to extraction efficiency or inhibition, adding another layer of assurance. Aligning sterility testing with modern manufacturing As cell and gene therapies continue to grow in scale and complexity, sterility testing is becoming a core element of manufacturing infrastructure. Rapid sterility testing approaches that deliver timely, reliable results are increasingly critical for supporting modern production timelines. Digital PCR-based solutions such as the Cyclus® dPCR Tool Box Bacteria Fungi reflect a shift toward sterility testing approaches that align more closely with the realities of modern ATMP production. By delivering faster, clearer sterility assessments, these methods help manufacturers balance patient safety with the need for timely product release, ensuring that when a therapy is ready, it can move forward with confidence. A rapid sterility testing solution designed for time-sensitive cell and gene therapies, the Cyclus® dPCR Tool Box Bacteria Fungi brings speed, precision, and confidence to ATMP quality control workflows. CREDIT: SARTORIUS