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Introduction: This article aims to identify best practices, improve risk controls, and aid regulatory agencies in developing guidance for environmental and biosafety risk assessment for commercial-scale cell and gene therapy manufacturing. Methods: A cross-functional team should start with hazard classification and testing requirements for materials used or generated by the process and process hazard characterization. Results: The team develops a safety profile of the process to mitigate risks, including: product biological contamination risk and process controls, including raw materials, facilities, operator and environmental controls, and method of detection;a technical review of the process to evaluate the operational and engineering controls;monitoring systems to mitigate the risk of failure and/or breach of the system, preventing the release of material to the facility or operator exposure;site sanitization strategy and facility containment measures, including engineering designs, air handling systems, spill containment measures, surface cleanability, waste flows, and decontamination practices;a review of site practices, including process, employee, material and waste flows, staff training, controlled access, operator gowning, and emergency response plans/measures. Discussion: The cross-functional team should regularly reconvene to provide solutions for enhanced process control, process life-cycle management, monitor assumptions, and track performance. The plan must be revised following any relevant failure event or process change. Conclusion: A risk assessment template is shared to bring to the reader's attention the complexity of commercial-scale manufacturing, areas to assess, potential questions to ask, and other pertinent parties who may input to the risk assessment.
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The Ortho trak-C immunoassay has recently established detection of the HCV core antigen as a viable indirect marker of HCV replication in clinical samples. In this study, trak-C is used to monitor HCV replication in three pre-clinical models: the cellular HCV replicon system, transient transfection of HCV genomes, and the murine Alb-uPa/SCID HCV infection model. All of these systems utilize full-length HCV genomes that direct the expression of core, facilitating its detection with monoclonal antibodies. When performed with purified protein, the assay detects HCV core with a lower limit of detection at 1.5pg, and exhibits linear detection up to 100pg. When assaying extracts prepared from Huh-7 clone 21-5 cells harboring a full-length HCV replicon, core is detectable from as few as 63 cell equivalents. The assay was used to determine the sensitivity of Huh 21-5 cells to the antiviral effects of interferon (IFN). Inhibition by IFN-alpha using core detection was comparable to that observed using branched-DNA (bDNA 3.0) detection of HCV RNA. Replication of transfected full-length HCV 1a Con1 genomes in Huh-7 cells was also detectable using the trak-C assay. Finally, in the transgenic murine HCV infection model, the course of viral amplification was detected from serum using trak-C with kinetics similar to those observed with RNA detection. Given its ease of use and the lack of requirement for RNA purification, the trak-C assay has several advantages over RNA-based methods of viral monitoring.