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<ns3:p> <ns3:bold>Background</ns3:bold> </ns3:p> <ns3:p>A strategic transition from animal-derived limulus amebocyte lysate (LAL) to synthetic recombinant Factor C (rFC) has become essential for securing a resilient, ethical, and future-proof global vaccine supply chain. Endotoxin testing is a required quality control step during vaccine and injectable drug manufacturing to ensure that products are free from bacterial toxins that could trigger serious inflammatory reactions in patients. Continued reliance on horseshoe crab–derived lysate exposes Gavi-supported vaccine programs to ecological risk, supply volatility, and increasing regulatory and reputational pressure. Because LAL is widely used not only for vaccines but also for biologics, injectable pharmaceuticals, and medical devices, the implications of this transition extend beyond the vaccine sector; however, this report focuses primarily on the Gavi-supported vaccine use case.</ns3:p> <ns3:p/> <ns3:p> <ns3:bold>Evidence base and methodology</ns3:bold> </ns3:p> <ns3:p>This report is based on a triangulated evidence base, including PATH-conducted manufacturer surveys, PATH in-house endotoxin testing (under a separate scope of work), and expert insights from a multi-stakeholder roundtable discussion held at the AFSA Conference in Bangkok in November 2025. Together, these inputs capture quantitative performance requirements, cost estimates, as well as operational, regulatory, and implementation considerations relevant to low- and middle-income country (LMIC) manufacturers transitioning from LAL to rFC. This evidence base reflects both the technical feasibility of rFC and the practical challenges manufacturers face when shifting away from long-established LAL-based testing workflows.</ns3:p> <ns3:p/> <ns3:p> <ns3:bold>Cost results</ns3:bold> </ns3:p> <ns3:p>Based on PATH analysis using an in-house endotoxin testing process across multiple mRNA vaccine formats, rFC adoption requires higher upfront capital investment for testing infrastructure, laboratory staff training, and regulatory bridging costs. We estimated that these costs are approximately ten times higher for rFC than for LAL (US$8,000 in annualized upfront costs per site, compared with US$800 for LAL). However, recurring costs for reagents, labor, and quality assurance are approximately 40 percent lower with rFC than with LAL. Our analysis estimates that if 10 million doses are produced and 200 tests are conducted per year, the cost per dose is US$0.0012 for LAL compared with US$0.0015 for rFC, accounting for annualized upfront costs and recurring testing costs. Importantly, while rFC requires greater initial investment, it offers meaningful operational efficiencies once implemented at scale. Our analysis found that rFC becomes cost-comparable at the high production volumes typical of human papillomavirus (HPV) vaccine and hepatitis B vaccine. Our modeling indicates that once implemented at scale, rFC reduces repeat testing by approximately 90 percent, improves batch-release predictability, and lowers long-term operating costs.</ns3:p> <ns3:p/> <ns3:p> <ns3:bold>Regulatory results</ns3:bold> </ns3:p> <ns3:p>The regulatory landscape has now shifted in favor of rFC adoption, as this method has been shown to be equivalent or superior to LAL in terms of analytical performance, including endotoxin detection sensitivity, specificity, and reduced susceptibility to assay interference (particularly from β-glucans). The rFC method has moved from an alternative method to a fully compendial standard, eliminating the need for burdensome dual validation and significantly reducing time, cost, and regulatory uncertainty for vaccine manufacturers. This shift is estimated to reduce validation-related labor and reagent consumption by approximately 50 percent. This regulatory milestone removes one of the most significant historical barriers to the broader adoption of animal-free endotoxin testing.</ns3:p> <ns3:p/> <ns3:p>Despite this progress, a clear “transition gap” remains for LMIC manufacturers—particularly micro, small, and medium enterprises (MSMEs)—driven by upfront capital requirements, limited technical capacity, and misalignment between manufacturers and national control laboratories. Without targeted intervention, these barriers risk slowing adoption precisely where global vaccine supply is most price-sensitive, even though PATH analysis indicates that rFC operating costs at scale approach parity with LAL (US$0.0015 versus US$0.0012 per unit throughput at high volume). Bridging this gap is essential to ensuring equitable access to modern endotoxin testing methods across all manufacturing settings, not only in high-income markets.</ns3:p> <ns3:p/> <ns3:p> <ns3:bold>Conclusion</ns3:bold> </ns3:p> <ns3:p>This report presents a four-phase, donor-enabled roadmap to close that gap (see Figure 1). Through market-shaping mechanisms, targeted technical enablement, regulatory alignment, and global scaling, the rFC transition can be accelerated in a way that is financially viable for LMIC manufacturers, compliant with global standards, and resilient to ecological and supply-chain shocks.</ns3:p> <ns3:p/> <ns3:p>The global health community has a rare opportunity: a one-time catalytic intervention that permanently decouples global vaccine safety testing from animal dependence, strengthens supply-chain resilience, and ensures that lifesaving vaccines for the world’s most vulnerable populations are produced using modern, sustainable, and scientifically superior methods.</ns3:p>