In the fields of pharmaceuticals and medical devices, ensuring the absence of bacterial endotoxin contamination in products is of paramount importance. Bacterial endotoxins, components of the lipopolysaccharides in the cell walls of Gram-negative bacteria, can trigger severe consequences like fever, shock, and multi-organ failure even at extremely low concentrations once they enter the human body. For instance, in the medical context, injectable drugs such as vaccines and antibiotics, as well as implantable medical devices like cardiac stents and artificial joints, can lead to disastrous outcomes if contaminated with endotoxins. Currently, TAL/LAL reagent testing serves as a crucial means to guarantee product safety. However, the acquisition of traditional TAL/LAL reagents is fraught with difficulties, which has spurred the research and development of genetically engineered synthetic substitutes.
I. Importance of TAL/LAL Reagent and Difficulties in Its Acquisition
1. Significance in detection: TAL/LAL reagent plays an irreplaceable role in bacterial endotoxin detection. Annually, a vast number of drugs, injectables, and human-implanted medical devices must undergo strict TAL/LAL reagent testing before market launch to ensure the absence of bacterial endotoxins and safeguard human safety. For example, during the production of COVID-19 vaccines, each batch was rigorously tested with TAL/LAL reagents. Medical devices in direct contact with blood, such as extracorporeal membrane oxygenation (ECMO) and dialysis equipment, also need to pass the bacterial endotoxin limit test. Typically, the bacterial endotoxin limit for these devices should be no higher than 0.5 EU/mL.
2. Problems in acquisition: The production of TAL/LAL reagents relies on blood collection from wild horseshoe crabs. Globally, around 500,000 horseshoe crabs are captured for blood extraction each year. Despite being released back into the wild after blood collection, their mortality rate remains as high as 10% - 30%. Overfishing, coupled with ecological degradation, environmental pollution, and habitat loss due to global warming, has caused a 70% decline in the population of North American horseshoe crabs. Moreover, due to the individual differences among horseshoe crabs, TAL/LAL reagents from different batches may vary, affecting the stability of detection results. Besides, TAL/LAL reagents contain other coagulation enzymes and are highly sensitive to pH value, temperature, and ionic strength, which may interfere with the coagulation cascade reaction and undermine the accuracy and reliability of experimental results. Consequently, the search for substitutes for TAL/LAL reagents has become extremely urgent.
II. Development Status of Genetically Engineered Synthetic Substitutes for TAL/LAL Reagent
With the advancement of technology, remarkable progress has been made in the development of genetically engineered synthetic substitutes for TAL/LAL reagents. Currently, several new substitutes have emerged, with recombinant TAL/LAL reagents (such as PSNG) and the recombinant factor C method (rFC) being the most prominent.
1. Recombinant TAL/LAL Reagent (PSNG): PSNG is a bacterial endotoxin quantitative detection reagent synthesized through genetic engineering technology. It fully mimics the enzymatic reaction of natural TAL/LAL reagent, consisting of recombinant factor C, recombinant factor B, and recombinant procoagulant enzyme, while eliminating factor G, which is prone to causing false positives. This makes PSNG independent of horseshoe crab blood, fulfilling the goal of sustainable development. Research has demonstrated that only when the three recombinant zymogens coexist can endotoxins activate protease activity. PSNG shows equivalence to LAL in endotoxin detection, and the protease activities of the three recombinant zymogens are significantly enhanced, amplifying the signal. In terms of detection speed, compared with typical kinetic chromogenic reagents, the detection time of PyroSmart NextGen™ (PSNG) is approximately halved. Unlike natural LAL reagents with inherent batch-to-batch variability, PSNG reagents exhibit excellent batch-to-batch consistency. Moreover, the addition of 1,3-β-D glucan does not interfere with PSNG's endotoxin detection, and a good correlation is maintained among different batches. When detecting various finished products, water, buffer solutions, etc., PSNG has an equal or lower non-interference dilution factor (NID) compared to LAL.
2. Recombinant Factor C Method (rFC): The recombinant factor C method involves the in vitro recombination of factor C, the first component of the horseshoe crab coagulation cascade reaction. Once activated by endotoxins, factor C cleaves the fluorescent substrate to generate a fluorescent complex, and the content of bacterial endotoxins can be quantified by detecting this complex. This method effectively avoids false positives caused by the bypass interference of factor G, enabling more accurate detection of endotoxin content and ensuring the biological safety of injectable products. Compared with the traditional kinetic LAL method, the recombinant factor C method has a single reaction mechanism. On July 26, 2024, the Microbiology Expert Committee of the United States Pharmacopeia Commission approved Chapter <86>: Bacterial Endotoxin Detection, allowing the use of the non-animal-derived recombinant factor C method (rFC) for endotoxin detection. The final text was released in November 2024 and officially came into effect in May 2025. In June 2021, Chapter 2.6.32 of the European Pharmacopoeia clearly designated the recombinant factor C method as an alternative for bacterial endotoxin detection. Notably, when using the recombinant factor C method for endotoxin detection, method validation is not required, and the detection procedure is consistent with the existing endotoxin detection method.
III. Advantages and Disadvantages of New Substitutes
I. Advantages
1. Sustainability: Both recombinant TAL/LAL reagents and the recombinant factor C method eliminate the reliance on horseshoe crab blood resources, significantly reducing the exploitation of endangered horseshoe crabs. This aligns perfectly with the concept of ecological sustainable development and resolves the severe damage to biological resources caused by the acquisition of traditional TAL/LAL reagents.
2. Accuracy and Specificity: By removing factor G, recombinant TAL/LAL reagent PSNG effectively prevents false positive results triggered by the activation of factor G by 1,3-β-D glucan. Similarly, the recombinant factor C method avoids the bypass interference of factor G, enabling more precise detection of endotoxin content. Compared with traditional TAL/LAL reagents, these new substitutes have witnessed remarkable improvements in detection accuracy and specificity.
3. Batch Stability: Unlike natural TAL/LAL reagents with inconsistent batch quality, genetically engineered synthetic substitutes like PSNG demonstrate outstanding batch-to-batch consistency. This characteristic is of great significance for ensuring the stability and reliability of detection results and facilitates quality control during the production process.
4. Detection Speed: Take PSNG as an example; its detection time is approximately half that of typical kinetic chromogenic reagents. This allows for quicker result acquisition, improves detection efficiency, and accelerates the production processes of drugs and medical devices.
II. Disadvantages
1. Cost Issues: In the early stages, rFC reagents were characterized by high production costs and expensive prices, which hindered their commercialization. Although technological advancements have led to cost reductions, compared with traditional TAL/LAL reagents that are relatively inexpensive and have dominated the market, genetically engineered synthetic substitutes still lack a cost advantage, which restricts their market promotion and application to some extent.
2. Technical Complexity: The production of genetically engineered synthetic substitutes for TAL/LAL reagents involves complex genetic engineering techniques. Every step, from gene recombination, protein expression, and purification to the final product preparation, requires professional technical personnel and sophisticated equipment. This places high demands on the technical strength and capital investment of production enterprises, increasing the difficulty of product research, development, and production.
3. Recognition Issues: Regulatory authorities in different countries have varying degrees of acceptance of new alternative methods. Although methods such as the recombinant factor C method have been recognized by certain pharmacopoeias, in some regions or fields, traditional TAL/LAL reagent detection methods are deeply ingrained. Therefore, it will take time and more practical verification for new substitutes to gain widespread recognition.
IV. Applications of New Substitutes
1. Pharmaceutical Industry: In pharmaceutical production, ensuring the absence of bacterial endotoxin residues in vaccines, injections, biological products, etc., is non-negotiable. New substitutes such as recombinant TAL/LAL reagents and the recombinant factor C method can be applied to the bacterial endotoxin detection of these drugs. For example, during the production of both traditional vaccines and new mRNA vaccines, each batch must undergo strict endotoxin testing. The high accuracy and specificity of new substitutes can better ensure vaccine quality. In the production of injectable antibiotics and other drugs, these new detection reagents can also be used to guarantee product safety and prevent serious medical accidents caused by endotoxin contamination.
2. Medical Device Industry: For medical devices that come into direct contact with blood or are implanted in the human body, such as ECMO, dialysis equipment, cardiac stents, and artificial joints, the bacterial endotoxin limit test is a crucial step to ensure safety. New substitute TAL/LAL reagents can be utilized for the detection of these medical devices during the production process and for finished product quality inspection. Take extracorporeal circulation medical devices as an example. Since they directly contact the patient's blood and are often used in combination with multiple devices, the endotoxins from different devices may accumulate. The application of new detection reagents can more accurately measure the endotoxin content, ensuring that products meet the bacterial endotoxin limit standards and reducing the risk to patients.
3. Other Fields: In the food and beverage industry, genetically engineered synthetic substitutes for TAL/LAL reagents can be explored for detecting bacterial endotoxins in products at risk of bacterial contamination, such as barreled drinking water and functional beverages, to ensure food safety. In the field of environmental monitoring, the high sensitivity and accuracy of new substitutes also hold potential for detecting bacterial endotoxins in samples like water bodies and soil, helping to more accurately assess environmental quality and ecological risks.
The development of genetically engineered synthetic substitutes for TAL/LAL reagents offers a new solution to the resource crisis and detection deficiencies of traditional TAL/LAL reagents. Although these new substitutes currently face challenges in terms of cost, technology, and recognition, with continuous technological progress and increasing market acceptance, they are expected to be more widely applied in pharmaceuticals, medical devices, and other related fields, playing a vital role in safeguarding public health and promoting ecological sustainable development.
