Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies.

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP<71>) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

Comparison of Five Commercial Molecular Assays for Mycoplasma Testing of Cellular Therapy Products.

Testing of cellular therapy products for Mycoplasma is a regulatory requirement by the United States Food and Drug Administration (FDA) to ensure the sterility and safety of the product prior to release for patient infusion. The risk of Mycoplasma contamination in cell culture is high. Gold standard testing follows USP 63 which requires a 28-day agar and broth cultivation method that is impractical for short shelf-life biologics. Several commercial molecular platforms have been marketed for faster raw material and product release testing; however, little performance data are available in the literature. In this study, we performed a proof-of-principle analysis to evaluate the performance of five commercial molecular assays, including the MycoSEQ Mycoplasma detection kit (Life Technologies), the MycoTOOL Mycoplasma real-time detection kit (Roche), the VenorGEM qOneStep kit (Minerva Biolabs), the ATCC universal Mycoplasma detection kit, and the Biofire Mycoplasma assay (bioMérieux Industry) using 10 cultured Mollicutes spp., with each at four log-fold dilutions (1,000 CFU/mL to 1 CFU/mL) in biological duplicates with three replicates per condition (n = 6) to assess limit of detection (LOD) and repeatability. Additional testing was performed in the presence of tumor infiltrating lymphocytes (TILs). Based on LOD alone, the Biofire Mycoplasma assay was most sensitive followed by the MycoSEQ and MycoTOOL which were comparable. We showed that not all assays were capable of meeting the ≤10 CFU/mL LOD to replace culture-based methods according to European and Japanese pharmacopeia standards. No assay interference was observed when testing in the presence of TILs.

Sterility Testing for Cellular Therapies: What Is the Role of the Clinical Microbiology Laboratory?

Sterility testing of cellular therapy products along with the associated environmental monitoring requirements for aseptic facilities, including compounding pharmacies, continues to impact clinical microbiology laboratories, as evidenced by the numerous discussions recurring on American Society for Microbiology Division C and ClinMicroNet listservs. This minireview provides an overview of this complex field of current good manufacturing practices (cGMP) based on biopharmaceutical industry standards and summarizes the compendial and alternative rapid microbial test methods available for product sterility and Mycoplasma testing. In addition, this minireview highlights major overarching regulatory requirements governing any laboratory performing product testing as regulated by the United States Food and Drug Administration (FDA). These requirements are different from the more familiar clinical requirements of the Clinical Laboratory Improvement Act of 1988 (CLIA '88), the College of American Pathologists (CAP), and the Joint Commission on Accreditation of Healthcare Organizations (JCAHO), all of which have no jurisdiction in this area. As the cellular therapy field continues to advance and an increasing number of medical centers participate in clinical trials of these novel therapies, it is critical that laboratories have a sound understanding of the major regulations and cGMP practices governing microbiological testing in the biopharmaceutical industry.

Comprehensive Evaluation of Compendial USP<71>, BacT/Alert Dual-T, and Bactec FX for Detection of Product Sterility Testing Contaminants.

The emergence of cell therapy programs in large academic centers has led to an increasing demand for clinical laboratories to assist with product sterility testing. Automated blood culture systems have shown promise as alternatives to the manual USP<71> compendial method, but current published data are limited by small organism test sets, particularly for molds. In 2015, failure of the Bactec FX system to detect mold contamination in two products prompted us to evaluate three test systems (compendial USP<71>, Bactec FX, and BacT/Alert Dual-T) over seven different culture combinations, using 118 challenge organisms representative of the NIH current good manufacturing practice (cGMP) environment. At <96 h and <144 h for bacterial and fungal detection, respectively, the compendial USP<71> method significantly outperformed the Bactec FX system (84.7% versus 64.4%; P = 0.0006) but not the BacT/Alert system at 32.5°C (78.8%; P = 0.3116). Extended incubation to 360 h with terminal visual inspection improved sensitivity, without a significant difference between compendial USP<71> and BacT/Alert testing (95.7% versus 89.0%; P = 0.0860); both systems were better than the Bactec FX system (71.2%; P < 0.0001 and P = 0.0003, respectively). The Bactec FX and BacT/Alert systems performed equivalently for 30 isolates derived from clinical bloodstream infections, confirming system optimization for clinical organisms rather than environmental contaminants. Paired Sabouraud dextrose agar (SDA) plates were always positive for fungi within the acceptable time frame. This study shows that the Bactec FX system is suboptimal for product sterility testing, and it provides strong data to support the use of BacT/Alert testing at 32.5°C paired with a supplemental SDA plate as an acceptable alternative to the compendial USP<71> method for product sterility testing.