{
  "abstract": "Background Mycoplasma contamination poses a persistent challenge in biopharmaceutical production, threatening both cell viability and patient safety. Conventional detection methods, such as culture and indicator cell assays, require up to 28 days for completion, delaying product release and increasing operational risk. These methods also depend heavily on consistent laboratory conditions, reducing reliability in detecting low-level contamination. Recent advances in molecular diagnostics have provided efficient alternatives, enabling sensitive and specific detection of mycoplasma DNA within a few hours. Recognized by major pharmacopeias, qPCR is increasingly adopted in the release testing of monoclonal antibodies, cell therapies, and viral vectors. Driven by the growing need for rapid and reliable contamination control, qPCR-based methods offer several advantages: short assay time, integration with automated workflows, and compatibility with complex sample matrices. In this study, we assess a validated qPCR assay developed under GMP-like conditions for its ability to detect over 250 mycoplasma species, supporting its application in regulated environments and high-throughput manufacturing settingsMethods To evaluate mycoplasma contamination with high sensitivity and specificity, we utilized a streamlined workflow integrating ACROBiosystems’ Automated Nucleic Acid Extraction instrumentation and Mycoplasma Sample Preparation and qPCR Quantitative Detection Kit, as outlined in the schematic overview (figure 1). The following section details the experimental procedures and control strategies employed throughout the assay.Results To validate the performance of the ACROBiosystems Mycoplasma Quantitative Detection Kit, we followed international guidelines outlined by EP <2.6.7>, USP <63>, JP, and the Eurofins Biopharma Product Testing framework for NAT comparability studies. The evaluation covered all essential parameters, including specificity, sensitivity, positive cut-off, and robustness, in alignment with regulatory expectations for nucleic acid-based mycoplasma detection.Conclusions This research workflow demonstrates that the ACROBiosystems qPCR-based mycoplasma detection method is a reliable alternative to conventional compendial assays, meeting key validation criteria outlined in EP <2.6.7>, USP <63>, and JP guidelines. The assay consistently achieved a detection limit of 10 CFU/mL across multiple mycoplasma strains, fulfilling the positive cut-off requirement of ≥95% detection in replicate testing. The method showed high specificity, with no cross-reactivity observed in unrelated cell lines or microbial strains, and demonstrated robustness against variations in reagent concentrations, extraction procedures, and qPCR instrumentation. In addition, the assay maintained performance in complex sample matrices, including those with high cell density and cryoprotectants such as DMSO.",
  "authors": [
    {
      "affiliations": [
        "ACROBiosystems, Newark, DE, USA"
      ],
      "name": "Lisa Chou"
    },
    {
      "affiliations": [
        "ACROBiosystems, Beijing, China"
      ],
      "name": "Xueying Sun"
    },
    {
      "affiliations": [
        "ACROBiosystems, Beijing, China"
      ],
      "name": "Jingjing Liu"
    },
    {
      "affiliations": [
        "ACROBiosystems, Beijing, China"
      ],
      "name": "Shumin Bai"
    },
    {
      "affiliations": [
        "ACROBiosystems, Beijing, China"
      ],
      "name": "Tingting Fan"
    },
    {
      "affiliations": [
        "ACROBiosystems, Beijing, China"
      ],
      "name": "Tianfu Zhang"
    },
    {
      "affiliations": [
        "ACROBiosystems, Beijing, China"
      ],
      "name": "Spencer Chiang"
    }
  ],
  "title": "1048 Rapid mycoplasma detection technologies: enhancing biopharmaceutical safety and efficiency",
  "uid": "e4600f6a-0039-5363-90a3-db6b3874cdac"
}