Rethinking vaccine manufacturing using cell-free systems: challenges and opportunities

Cell-free protein synthesis (CFPS) has emerged as an attractive technology for decentralized manufacturing of proteins, offering the ability to decouple lysate production from protein synthesis, enabling flexible batch sizing, rapid deployment, and compatibility with a broad range of biological targets. Despite recent achievements, including the first GMP-compliant CFPS manufacturing run at 4,500 L scale, significant technical, analytical, and regulatory challenges remain before CFPS can be routinely adopted for vaccine manufacturing. This review examines the key challenges and opportunities across the CFPS workflow, including the need for robust lysate characterization strategies, definition of the CQAs and Critical Process Parameters that govern lysate consistency, and downstream processing considerations unique to CFPS, such as the higher burden of host cell proteins, ionic complexity, membrane-derived vesicle impurities, and endotoxin management. The successful translation of CFPS to vaccine manufacturing requires a fundamental shift from rapid prototyping toward manufacturing-ready development. This will require more rigorous analytical characterization, early regulatory engagement, and a clear deployment strategy. Rather than competing with established cell-based platforms at scale, CFPS offers its greatest value in speed, flexibility, and decentralization, which are all critical factors in vaccine responsiveness.