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General
The Sustainability Scorecard for Endotoxin Testing measures and tracks the adoption of synthetic alternatives to horseshoe crab blood for safety testing drugs and vaccines. The Scorecard reports on the progress of the world’s largest pharmaceutical companies to transition away from using horseshoe crab blood for safety testing injectable medicines and vaccines.
On November 1, 2024, the U.S. Pharmacopeia (USP) adopted and published endotoxin (contaminant) testing guidelines for the use of synthetic alternatives in lieu of horseshoe crab-derived Limulus amoebocyte lysate (LAL).
Based on USP’s guidance, synthetic alternatives are now considered equivalent; in essence, creating a level playing field between the gold standard LAL and synthetic alternatives. Now pharmaceutical companies have the opportunity to do the right thing for patient safety, and for the protection of horseshoe crabs and the ecosystems that rely on this critical species.
“Endotoxin testing is a critical step in ensuring the quality and safety of many sterile pharmaceutical products. Chapter <86> includes methods that use both recombinant cascade (rCR) and recombinant Factor C (rFC) reagents and provides information for manufacturers to incorporate these reagents into their quality testing…The addition of Chapter <86> to the US Pharmacopeia is in line with USP’s commitment to expanding the use of animal-free methods and materials.” – USP News Release, November 1, 2024
The increasing demand for vaccines, injectable medications, and infusion therapies to treat cancers, infectious diseases, diabetes, obesity, autoimmune conditions and other illnesses has led to a corresponding surge in the harvesting of horseshoe crabs for endotoxin (contaminant) testing. The adoption of synthetic alternatives to the blood of this declining species is crucial for ensuring a reliable and sustainable supply of endotoxin testing solutions without compromising patient safety.
Just as advances in biotechnology are fueling a revolution in drug discovery and development, biotechnology has also provided a sustainable and scalable safety testing solution in the form of synthetic alternatives to LAL. Synthetic alternatives are a proven solution that is fully scalable to meet rising product demand while supporting industry’s commitment to the 3Rs in animal testing (Replace, Reduce, Refine).
The Scorecard is a joint initiative of Revive & Restore, the Horseshoe Crab Recovery Coalition (HCRC), and the Center for Biological Diversity.
- Revive & Restore is the leading wildlife conservation organization promoting the incorporation of biotechnologies into standard conservation practice. The Sausalito, California nonprofit was formed in 2012 with the idea that 21st century biotechnology can and should be used to enhance genetic diversity, build disease resistance, and facilitate adaptation. Its mission is to enhance biodiversity through the genetic rescue of endangered and extinct species.
- The Horseshoe Crab Recovery Coalition (HCRC) is composed of more than 50 biomedical and conservation groups dedicated to ensuring the future of the American Horseshoe Crab. In addition to the Center for Biological Diversity and Revive & Restore, multiple Audubon societies, the National Wildlife Federation, the American Littoral Society, the American Bird Conservancy, Physicians’ Committee for Responsible Medicine, the Humane Society of the United States and the Humane Society International are among the coalition’s leading partners. Since 2018, the group has led science-based campaigns aimed at halting years of decline in horseshoe crab populations and to fully restore them by 2030.
- The Center for Biological Diversity believes that the welfare of human beings is deeply linked to nature. Because diversity has intrinsic value, and because its loss impoverishes society, the Center works to secure a future for all species, great and small, hovering on the brink of extinction. The Center uses science, law and creative media to protect the lands, waters and climate that species need to survive.
To better understand the industry’s progress in adopting more sustainable endotoxin testing solutions, we have developed a survey for the 50 largest pharmaceutical companies based on 2023 total revenue. The survey asks company representatives to indicate which provided statements are true about their company’s safety testing methodologies and their plans to transition to using synthetic alternatives.
Each potentially true statement is worth a number of points. A company’s total number of points corresponds to a sustainability rating of 1-5 horseshoe crabs. The more points, the higher the sustainability rating.
In December 2024, the survey was sent to business representatives at the 50 largest pharmaceutical companies in the world. Completed surveys were requested by February 1, 2025. We anticipate that this first survey will initiate an ongoing conversation with the biomedical community as more companies transition to synthetic alternatives for safety testing.
| POINT SYSTEM | SUSTAINABILITY RATING |
| 1 – 20 POINTS |  |
| 21 – 40 POINTS |  |
| 41 – 60 POINTS |  |
| 61 – 80 POINTS |  |
| 81 – 100 POINTS |  |
The survey is organized into five categories. Each category describes a group of potential steps taken by companies to transition away from using horseshoe crab blood for drug and vaccine safety testing. Companies with points in multiple categories are further along in their transition to using synthetic alternatives.
- Companies earning points in Category 1 no longer use TAL, derived from the (already endangered) Asian Horseshoe Crab
- Companies earning points in Category 2 have publicly acknowledged and/or published data about the benefits of removing LAL from research and manufacturing processes.
- Companies earning points in Category 3 are significantly reducing the use of LAL (by at least 80%) in manufacturing processes via transition to synthetic alternatives for water testing and/or via microfluidics.
- Companies earning points in Category 4 are working to adopt, or have already adopted, synthetic alternatives for safety testing in the manufacturing of new applicable products.
- Companies earning points in Category 5 are working to adopt, or have already adopted, synthetic alternatives for safety testing in the manufacturing of legacy (previously registered) products.
Starting in Spring 2025, companies that do not submit a survey will appear on the Scorecard with zero points.
Yes. We look forward to each company’s score improving as they continue to remove horseshoe crabs from their drug and vaccine manufacturing processes. We will update the Scorecard regularly as we learn about additional ways that companies are making the transition to synthetic alternatives.
The American Horseshoe Crab is currently listed as a “vulnerable” species by the International Union for the Conservation of Nature (IUCN) and its designation as a “U.S. Endangered Species” is under consideration by the National Marine Fisheries Services (NOAA Fisheries).
The Atlantic States Marine Fisheries Commission (ASMFC) estimates mortality for bled horseshoe crabs at 15 percent, but independent studies show mortality could be as high as 30 percent, with additional effects beyond mortality that impact the population’s ability to spawn for one year. Harvesting horseshoe crabs for their blood now exceeds the bait harvest, with more than 1.1 million crabs being bled annually for pharmaceutical purposes. American Horseshoe Crabs play a critical role in coastal ecosystems in the United States, including the species’ vital importance to migratory shorebirds.
What are the benefits for companies to adopt synthetic alternatives for endotoxin testing?
Beyond its sustainability benefits, synthetic alternatives offer several advantages over traditional LAL tests:
- Equivalent Efficacy: synthetic solutions demonstrate equivalent effectiveness in detecting bacterial contaminants.
- Reduced False Positives: synthetic solutions eliminate the potential for false positives and elevated readouts associated with variations in horseshoe crab blood.
- Scalability and Consistency: synthetic solutions avoid supply chain constraints and ensure consistent results, unlike the variable nature of LAL tests.
- Alignment with 3Rs: Adopting synthetic alternatives align with the pharmaceutical industry’s commitment to the 3Rs (Replacement, Reduction, and Refinement) of animal testing.
The transition would happen in stages. Here is a potential staged approach:
- Prioritize Water Testing: Implement synthetic alternatives for lab water testing. Lab water testing has been shown to utilize approximately 80% of the use of LAL in large pharmaceutical manufacturing processes.
- New Drug/Vaccine Development: Integrate synthetic alternatives into the development pipeline for testing new applicable drugs.
- Legacy Drug/Vaccine Application: Once established, consider applying synthetic alternatives to testing legacy (previously registered) drugs/vaccines.
For Pharma R&D and Production
Yes, rCRs are known to be cost-equivalent to LAL. rFC is less expensive because there is only 1 recombinant protein involved instead of 3 in the rCRs.
The European Pharmacopoeia has recognized these advantages and accepted rFC as an equivalent method in chapter 2.6.32 since 2020. The US Pharmacopeia will officially recognize rFC as an equivalent method in May 2025. Chapter 86 has already been published for early adoption.
Multiple peer-reviewed studies and regulatory evaluations have demonstrated that synthetic alternatives, particularly recombinant Factor C (rFC), compare favorably to traditional LAL:
- Sensitivity: rFC is equally or more sensitive than LAL, with detection limits as low as 0.001 EU/mL
- Specificity: rFC has superior specificity for endotoxins, as it contains only Factor C rather than multiple factors in LAL that can react with (1,3)-β-D-glucans and other non-endotoxin substances
- Consistency: Synthetic alternatives show lower variability (coefficient of variation typically <10% compared to 15-30% for LAL) between lots since they are produced under controlled recombinant conditions rather than being harvested from wild animals with natural biological variability
- Robustness: Studies have shown that synthetic alternatives are less susceptible to interference from complex pharmaceutical formulations
The regulatory landscape has evolved significantly:
- The FDA has stated that the agency would accept synthetic alternatives since they issued the 2012 Guidelines; rFC has been approved for use in at least 75 countries.
- The US Pharmacopeia will officially recognize rFC as an equivalent method in May 2025. Chapter 86 has already been published for early adoption.
- As of 2024, synthetic alternatives are included in the PhEur, British, and Eurasian Pharmacopeias; China, Japan, and Korea have guidance chapters as well.
Timeline:
- Current status: Synthetic alternatives are accepted globally but may require additional validation in some markets
- Europe recently proposed combining 2.6.32 back into 2.6.14 which would pave the way for full global harmonization
Companies typically need 9 months to transition if there is no regulatory impact (e.g. water and new products). Most likely, the transition will take multiple years for legacy products.
Companies that have successfully transitioned report that maintaining open communication channels with both suppliers and regulatory agencies throughout the process is critical for preventing disruptions.
A successful transition requires a staged implementation approach:
- Risk-based implementation: Begin with lower-risk products or those with stable manufacturing processes
- Contingency planning: Develop specific protocols for handling any unexpected testing issues
- Maintain dual inventory: Keep sufficient LAL reagent inventory during the transition period (typically 12-18 months)
- Supplier qualification: Qualify multiple suppliers of synthetic alternatives to mitigate single-source risks
- Validation master plan: Develop a comprehensive validation strategy that accounts for different product types
- Staff training: Implement robust training programs before official transition
- Regulatory strategy: Prepare change control documentation and regulatory submissions in advance
Validation requirements vary by regulatory region but typically include:
- Method validation: Demonstrating appropriate sensitivity, specificity, accuracy, precision, and robustness. Validation data can typically be leveraged from the reagent supplier.
- Product-specific verification: Testing for potential product interference with the new method
- Comparative studies: Side-by-side testing with traditional LAL to demonstrate equivalence. This step may be needed at the request of a health authority, but industry experience to date is that it has not been needed. If needed, a simple 3 lot comparison of results with LAL to recombinant is sufficient.
- Method transfer: Validating the method across different testing sites if applicable
Resource requirements:
- Timeline: 1-3 months for initial validation of the method
- Personnel: Typically requires 1-2 dedicated analysts and QA resources
- Sample requirements: 30-50 samples per product type for comprehensive validation (same as LAL)
- Documentation: Approximately 100-200 pages of validation documentation per product type. With the exception of initial validation and new methods/SOPs, this is the same as LAL.
- Cost: <$10,000 if no new equipment is involved. The cost of regulatory filings is unknown.
Many suppliers of synthetic alternatives now provide validation support packages that significantly reduce this burden, including validation protocols, statistical analysis tools, and technical support.
Companies that have fully implemented synthetic alternatives typically report overall efficiency improvements of 20-30% in their QC testing workflows.
Synthetic alternatives offer several efficiency advantages:
- Reduced preparation time: Synthetic reagents typically require less preparation time (15-30 minutes less) than LAL (e.g. ready-to-use plates and cartridge formats)
- Faster time-to-result: Comparable to LAL (15-90 mins.), recombinants are 15-60 mins depending on assay sensitivity and equipment.
- Automation compatibility: Synthetic alternatives are highly compatible with automated systems and robotics, allowing for higher throughput testing
- Simplified sample preparation: Less interference means simpler sample preparation in many cases
- Broader testing conditions: Some synthetic alternatives can operate under a wider range of pH and temperature conditions
- Extended calibration stability: Many synthetic systems require less frequent calibration
Financial modeling indicates that companies remaining fully dependent on LAL face potential cost increases of 40-60% over the next decade, not including the significant risks of supply disruptions.
Continuing dependence on horseshoe crab-derived LAL presents several significant financial risks:
- Price volatility: LAL costs have increased 3-5% annually and are projected to accelerate to 7-10% as supply constraints worsen
- Supply interruptions: Limited harvesting seasons and declining populations have led to allocation situations where manufacturers receive only partial orders
- Geographic restrictions: Increasing harvesting regulations in key regions may further constrain supply
- Quality inconsistencies: Greater biological variability as suppliers are forced to harvest from different populations or at suboptimal times
- Regulatory risks: Growing probability of stricter regulations on endangered species-derived materials in pharmaceutical testing
- Emergency sourcing costs: Companies have reported paying 2-3x normal prices during supply shortages
- Inventory carrying costs: The need to maintain larger safety stocks increases working capital requirements
- Release delays: Testing backlogs during supply constraints can delay product release by 2-4 weeks
- Reputation damage: Growing investor and consumer awareness of horseshoe crab conservation issues poses brand risks. TAL sourced from the already endangered Asian Horseshoe Crab is a cautionary tale in supply chain stability. rFC was invented in 1995 in response to dwindling wild harvests of Asian Horseshoe crabs.
The integration process typically requires 1-2 weeks of validation and verification per automated system, with suppliers providing on-site technical support for the transition.
Integration with existing automation platforms has significantly improved in recent years:
- All synthetic alternatives are compatible with major equipment, software, and automation platforms.
- Validation packages: Suppliers provide specific validation protocols for each major automated platform
- Method parameters: Most systems require only minor adjustments to existing parameters rather than complete reconfiguration
- Data systems: Synthetic alternatives use comparable data formats, requiring minimal LIMS or data management system changes
- Throughput: No reduction in throughput capacity when transitioning; many companies report increased throughput
- Dual-use capability: Most modern systems can run both LAL and synthetic tests, allowing for parallel testing during transition
- Software updates: Major automation suppliers now provide specific software modules for synthetic alternatives
- Cartridge systems: Pre-loaded cartridge-based systems are available for synthetic alternatives, maintaining the convenience of similar LAL systems
Synthetic alternatives generally offer advantages in stability and storage requirements:
- Shelf life: Most synthetic alternatives have 24-36 month shelf lives compared to 12-18 months for LAL
- Storage temperature: While LAL typically requires refrigeration (2-8°C), some synthetic alternatives offer room temperature stability (15-30°C) for short periods and refrigerated storage for long-term
- Freeze-thaw stability: Synthetic alternatives generally withstand 3-5 freeze-thaw cycles without significant loss of activity, compared to 1-2 cycles for LAL
- Reconstituted stability: rFCs are liquid so there is no reconstitution, and are stable per the kit expirations. rCRs have short expirations once reconstituted, similar to LAL.
- Working solution stability: Working solutions of synthetic alternatives maintain calibration stability for 8-24 hours versus 2-4 hours for many LAL formulations
- Shipping conditions: Comparable (domestic) shipping processes as LAL.
- Environmental robustness: Synthetic alternatives show less sensitivity to environmental factors like ambient light and vibration
- Consistency between lots: Less variability in stability profiles between manufacturing lots
These enhanced stability characteristics translate into approximately 15-25% reduction in waste due to expired reagents and greater flexibility in laboratory operations. These are key cost improvement drivers.