Combining a Respiration-Based Method with Universal qPCR for 3-Day Rapid Sterility Testing

In this article, we present a rapid sterility method with a time to detection of 3 days and a limit of detection (LOD) of 1 CFU for testing both cell-therapy drug products and <i>E. coli</i> DNA plasmid preparations. The new method combines the automated rapid sterility test system BacT/ALERT (bioMérieux) as an enrichment step with PCR detection of viable microorganisms via the ATMP sterile release qPCR assay (Sartorius). Sample preparation protocols for non-filterable cell-therapy drug products and cell-free <i>E. coli</i> DNA plasmid preparations were developed; these include removal of contaminating DNA and PCR inhibitors and the use of a physical lysis step. A validation of the rapid sterility method using the BacT/ALERT system is presented and pre-validation experiments for the full assay are described.