Sterility testing in the era of decentralised cell therapy: Challenges, innovations, and future directions

Chimeric antigen receptor (CAR)-T cell therapies have transformed the treatment of haematological malignancies, yet patients with aggressive, time-sensitive disease remain vulnerable to manufacturing-related treatment delays. Current gold-standard sterility testing under United States Pharmacopoeia (USP) <71> requires a 14-day culture period that is structurally incompatible with the clinical timelines of short shelf-life cell therapy products, positioning sterility assurance as the principal bottleneck to decentralised CAR-T manufacturing. Rapid sterility testing methods have emerged as promising alternatives that may resolve this constraint and expand patient access to treatment.

We undertook a narrative review of the literature on sterility testing in cell therapy manufacturing to characterise current methodologies and the translational challenges of microbial detection in cell therapy products. Relevant publications were identified through searches of several biomedical databases, including MEDLINE, EMBASE, PubMed and Cochrane CENTRAL, supplemented by review of reference lists in relevant articles. Eligible studies reported original data relating to sterility testing in cell therapy products or addressed regulatory, infrastructural or procedural considerations relevant to GMP-compliant or decentralised manufacturing environments.

ATP bioluminescence, solid phase cytometry, and PCR-based assays targeting conserved 16S/18S rRNA sequences have emerged as rapid alternatives to compendial methods, delivering substantially shorter turnaround times. However, four translational barriers continue to preclude their widespread integration. First, analytical validation requirements demand matrix-specific performance data generated under actual CAR-T manufacturing conditions. Second, the operational infrastructure demands of decentralised sites, spanning personnel training, equipment qualification, and documentation capacity, often makes such data difficult to generate. Third, regulatory acceptability remains inconsistent across jurisdictions with no harmonised pathway for alternative method approval. Fourth, clinical risk governance is hampered by the absence of standardised protocols for managing discordant results that would support conditional release decisions. In response, we propose a structured three-phase validation framework for the conditional release of CAR-T products using rapid sterility assays, progressing from analytical qualification through bridging and equivalency to conditional release implementation, with defined decision gates at each phase transition.

Validating and integrating rapid sterility testing into decentralised CAR-T manufacturing workflows offers a credible route to shorter production lead times and broader access to CAR-T cell therapy. Significant barriers remain, however, most notably regulatory fragmentation and a paucity of real-world validation data that currently preclude widespread adoption. The three-phase validation framework proposed in this review provides a structured pathway for conditional release of CAR-T products using rapid sterility assays, progressing from analytical qualification through bridging and equivalency to conditional release implementation, with defined decision gates at each phase transition. Realising the full potential of this framework requires the field to treat regulatory harmonisation and prospective assay validation as jointly necessary conditions for equitable access to cell and gene therapy.