Insight

The development of pharmaceutical manufacturing processes involves systematic optimization of cell sources, culture medium compositions and bioreactor parameters to maximize the yield and production efficiency of target products. Among various expression systems, Escherichia coli has emerged as a cost-effective production platform for recombinant proteins due to its rapid proliferation rate, high product yield and convenient genetic manipulation. Nevertheless, regardless of the expression system adopted, endotoxin removal in downstream purification remains a major challenge in biopharmaceutical manufacturing.

Endotoxins derive from the outer membrane of Gram-negative bacteria, and their lipid A moiety possesses potent pyrogenic activity. Even extremely low doses can trigger severe inflammatory responses in vivo. Characterized by high thermal stability and easy binding affinity to target biomolecules, endotoxins are notoriously difficult to eliminate. Accordingly, establishing sensitive and reliable endotoxin detection methods alongside efficient removal strategies is essential to guarantee pharmaceutical product safety.

The rabbit pyrogen test is the earliest conventional method applied for endotoxin detection, which is based on the principle that endotoxins can induce febrile reactions in rabbits similar to those occurring in humans. In this assay, test samples are injected into live rabbits; a temperature rise exceeding 0.5 °C within 180 minutes post-injection is defined as a positive pyrogen reaction.

Established in 1912, this method features a limit of detection (LOD) of 0.5 EU/mL and was deemed sufficiently sensitive with intuitive and credible results in the early stage. However, with technological advances and rising ethical awareness, its drawbacks have become increasingly prominent. This approach requires substantial sample consumption and numerous experimental animals, while its sensitivity and accuracy fail to meet modern testing standards, and it also faces growing ethical concerns over animal welfare. Although it is still adopted by a small number of institutions in certain regions such as Japan, the scientific community has widely shifted toward more advanced and ethically compliant in vitro detection alternatives.

Developed in the 1960s, the Limulus Amebocyte Lysate (LAL) assay is an animal-free in vitro detection technique utilizing extracts from horseshoe crab blood. It is mainly categorized into three formats: gel-clot method, chromogenic method and turbidimetric method.

The gel-clot method judges endotoxin presence by observing stable gel formation after sample-reagent incubation, with an LOD ranging from 0.03 to 0.06 EU/mL. The chromogenic and turbidimetric methods are photometric assays requiring optical readers, which quantify endotoxin concentrations by measuring yellow color intensity released from chromogenic substrates or solution turbidity variations respectively. All LAL-based detections rely on the endotoxin-induced activation of Factor C in horseshoe crab blood, which initiates a coagulation cascade reaction and ultimately leads to gelation, color development or turbidity elevation.

Recognized as the official endotoxin testing method for pharmaceuticals and medical devices by regulatory authorities including the US FDA, LAL assay delivers higher sensitivity than the rabbit pyrogen test. Even so, it still has inherent limitations. Matrix interference caused by buffer components and surfactants in samples may lead to false-negative outcomes known as low endotoxin recovery. In addition, (1→3)-β-D-glucan can trigger false-positive results via activating the Factor G pathway. To reduce reliance on natural horseshoe crab resources, recombinant Factor C-based detection technologies have been developed in recent years.

The recombinant Factor C (rFC) assay is an in vitro endotoxin detection technique based on synthetic recombinant proteins. Its core principle lies in the specific activation of DNA-cloned recombinant Factor C protein by endotoxins, which further cleaves fluorescent substrates to generate detectable fluorescent signals measurable at 380/440 nm, and the fluorescence intensity is positively correlated with endotoxin concentration.

This method covers a detection range of 0.05–500 EU/mL. Since it excludes the glucan-induced activation pathway involved in LAL assays, it effectively eliminates false-positive results caused by glucan interference. Despite its excellent specificity and sensitivity under laboratory conditions, rFC detection is susceptible to environmental contamination in on-site applications, and its testing performance is highly dependent on the stability of recombinant proteins. It is comparable to LAL assay in testing costs, yet mandatory fluorescence reading equipment restricts its practical application scope and overall detection efficiency.

Continuously optimized since 1995, the monocyte activation test (MAT) serves as a viable alternative to the rabbit pyrogen test for in vitro pyrogen detection in humans. This technique co-cultures test samples with human monocytes to simulate in vivo pyrogenic responses. Upon exposure to endotoxins and other pyrogens, monocytes are activated and secrete pro-inflammatory cytokines such as interleukin-1β and interleukin-6.

Commercially available test kits combined with cryopreserved human monocytes are commonly used in practical operations, and cytokine levels are quantified via enzyme-linked immunosorbent assay (ELISA). Cytokines bind to primary antibodies and subsequently react with horseradish peroxidase-conjugated secondary antibodies to catalyze substrate color development, and absorbance values are measured at 450 nm for quantitative analysis. With an LOD of 10 EU/mL, MAT stands out for its capability to simultaneously detect endotoxins and non-endotoxin pyrogens without experimental animal usage. Nevertheless, limited sources of human monocytes and the short in vitro lifespan of viable cells (less than 2 hours) impair the consistency and reproducibility of detection results.

The bovine whole blood assay is an endotoxin detection technology based on animal whole blood incubation. Test samples are co-incubated with bovine whole blood, and endotoxins stimulate leukocytes in blood to synthesize prostaglandin E2, whose production level is proportional to endotoxin concentration.

This assay achieves an LOD of 0.25 EU/mL, close to the human endotoxin exposure threshold of 0.30 EU/mL, showing great potential to replace LAL and rabbit pyrogen tests in terms of detection accuracy. It also features simple operating procedures and minimal sample pretreatment with favorable practical applicability. However, it has multiple constraints: low endotoxin recovery may occur due to reagent interference, non-specific binding between endotoxins and blood cell receptors, or endotoxin masking effects; experimental calf whole blood is in short supply for large-scale preparation; furthermore, cultural and religious taboos in certain regions prohibit the use of bovine blood, further limiting its popularization and application.

Electrochemical biosensors represent a mainstream advanced technique for endotoxin detection, most of which are constructed based on electrochemical impedance spectroscopy. Specific endotoxin recognition proteins such as endotoxin-neutralizing proteins are immobilized on electrode surfaces; the binding reaction between endotoxins and immobilized proteins induces interfacial impedance changes to realize quantitative detection.

For instance, sensors fabricated with gold electrodes and recombinant TLR4-MD-2 protein complexes achieve an ultra-low LOD of 0.0002 EU/mL with outstanding sensitivity and specificity. Other newly developed sensors including polymyxin B (PMB)-modified porous silicon membrane sensors and peptide-functionalized gold electrode sensors cover a detection range of 0.001–18 EU/mL. Apart from impedance-based methods, amperometric and potentiometric methods are also widely adopted. Amperometric assays are easy to operate and cost-effective, while potentiometric assays support real-time monitoring despite relatively lower sensitivity. In general, electrochemical methods outperform traditional biological assays in sensitivity, detection speed and economic efficiency, yet they require sophisticated testing equipment and stringent experimental conditions as well as professional operating personnel.

Optical biosensing technologies provide high-sensitivity analytical tools for endotoxin quantification. Liquid crystal-based optical biosensors adopt endotoxin-specific single-stranded DNA aptamers as recognition probes, attaining an LOD of 5.5 EU/mL and exhibiting favorable linear responses within the range of 0.05–1000 EU/mL with high specificity and recovery rate.

Major categories of optical detection technologies include luminescence-based assays, surface plasmon resonance assays and electrochemiluminescence assays. All these methods realize rapid and visual endotoxin detection by capturing visible or instrument-readable variations in optical signals.

Fluorescence and luminescence detection technologies are characterized by ultra-high sensitivity and rapid response speed in endotoxin testing. Bioluminescence assays follow the core principle of LAL assays while adopting mutant firefly luciferase and pNA substrates, achieving an LOD as low as 0.0005 EU/mL with a reaction duration of merely 15 minutes, which is far more efficient than conventional gel-clot methods.

In fluorescence detection, lipophilic fluorescent dyes such as BODIPY undergo fluorescence quenching in the presence of endotoxins, and precise endotoxin quantification is realized by monitoring fluorescence intensity changes with excellent linear correlation (R² > 0.99). Additionally, Alexa Fluor-labeled fluorescent endotoxin probes and nanomaterial-based fluorescent sensing platforms are applied for endotoxin capture and quantification with an LOD of 10 EU/mL. These technologies are ideal for rapid endotoxin analysis in biological solutions due to fast response, high sensitivity and reliable quantitative performance.

Surface plasmon resonance (SPR) and mass-based sensing technologies diversify the technical routes for endotoxin detection. Portable smartphone-based detection platforms and U-shaped optical fiber probe SPR sensors have been developed to detect refractive index variations induced by endotoxin binding, achieving an LOD of 40 EU/mL within approximately 1 hour.

Among mass-based sensing techniques, electromagnetic piezoelectric acoustic sensors utilize polyethylene glycol-functionalized polymyxin B as recognition elements to achieve real-time plasma endotoxin monitoring within the detection range of 30–60 EU/mL. Bacteriophage-modified magnetoelastic sensors detect bacterial concentrations via resonant frequency shifts with a sensitivity of 500 CFU/mL and a total detection time of around 30 minutes, featuring low costs and wireless measurement capability, yet suffering from drawbacks including difficult surface regeneration and severe non-specific adsorption interference.

Endotoxin detection is an indispensable and core link to ensure biopharmaceutical product safety. As the mainstream quality control standard for decades, conventional LAL assays have laid a solid foundation for industrial endotoxin monitoring by virtue of mature standardization and superior sensitivity. Nevertheless, its dependence on natural horseshoe crab resources and susceptibility to matrix interference have driven the continuous exploration of innovative detection solutions across the industry.

Emerging biosensing and physicochemical detection technologies are accelerating the technological iteration of endotoxin testing. Recombinant Factor C-based biological assays show promising prospects in breaking reliance on natural biological resources and improving detection specificity. Meanwhile, diversified electrochemical and optical biosensing technologies are advancing endotoxin detection toward real-time monitoring, portability and high-throughput screening, laying a solid technical foundation for in-situ online monitoring throughout the entire biopharmaceutical production process.