Supramolecular assembly of micellar aggregates is the basis of low endotoxin recovery (LER) in a drug formulation that can be resolved by a whole blood assay.

Lipopolysaccharide (LPS, endotoxin) is ubiquitous and represents a harmful contaminant of pharmaceutical compounds, recombinant biologicals and drug products. The pyrogen can induce severe immune responses and pathology in vitro and in vivo. Health authorities require strict control of endotoxin in parenteral drugs. However, for research and pre-clinical compound analysis, endotoxin testing is not a required quality control, which may cause potential drawbacks in the translational pipeline. Endotoxin testing is usually performed by the Limulus amebocyte lysate (LAL) assay, which is hampered by the so-called low endotoxin recovery (LER) effect when certain drug formulations are tested. A comprehensive study including structural, biophysical, and biological analyses was conducted to identify LER root cause for phosphate- and polysorbate-containing parenteral drug products. LPS in water showed extended ribbon-like aggregate structures. In placebo (formulation buffer without drug) and in drug product (drug in formulation buffer), a reaggregation of LPS into a network of interlinked micelles with hidden head group charges, and a strong reduction of the negative surface potential was observed. The non-accessibility of the LPS backbone has a direct impact leading (i) to a loss of activation of the LAL-cascade, (ii) reduced activation of the TLR4/MD-2 receptor system, and (iii) increased survival in a mouse model of endotoxemia. These data provide a structure-based explanation of the LER-underlying mechanisms. A human whole blood assay is shown to resolve LER and detect the pyrogenic activity of endotoxin with high sensitivity. This may open new test options to improve quality control in drug development and drug safety.

Heparin enables the reliable detection of endotoxin in human serum samples using the Limulus amebocyte lysate assay.

The determination of lipopolysaccharide (endotoxin) in serum or plasma samples using Limulus amebocyte lysate (LAL)-based assays is currently not sufficiently reliable in clinical diagnostics due to numerous interfering factors that strongly reduce the recovery of LPS in clinical samples. The specific plasma components responsible for the endotoxin neutralizing capacity of human blood remain to be identified. There are indications that certain endotoxin-neutralizing proteins or peptides, which are part of the host defense peptides/proteins of the innate immune system may be responsible for this effect. Based on our finding that several antimicrobial peptides can be neutralized by the polyanion heparin, we developed a heparin-containing diluent for serum and plasma samples, which enables reliable quantification of LPS measurement in clinical samples using the LAL assay. In a preclinical study involving 40 donors, this improved protocol yielded an over eightfold increase in LPS recovery in serum samples, as compared to the standard protocol. This modified protocol of sample pretreatment could make LPS measurement a valuable tool in medical diagnostics.

Barriers to the Use of Recombinant Bacterial Endotoxins Test Methods in Parenteral Drug, Vaccine and Device Safety Testing.

The Bacterial Endotoxins Test (BET) is a critical safety test that is used to detect bacterial endotoxins, which are the major contributor to fever-inducing contamination risks known as pyrogens. All parenteral therapies, including every lot of injected drugs, vaccines, medical devices, must be tested for pyrogens to ensure patient safety. Bacterial endotoxins test methods were developed as a highly sensitive detection method for bacterial endotoxins, after the discovery of a clotting cascade in horseshoe crab blood. However, horseshoe crab species are limited to some inshore coastal habitats along the Atlantic coast of the USA and others throughout Asia. Fully functional horseshoe crab clotting factors can be manufactured via recombinant protein production, and several BET methods featuring recombinant horseshoe crab proteins have now been developed for commercial use. Recombinant Bacterial Endotoxins Test (rBET) methods based on the use of recombinant Factor C (rFC) were established in the European Pharmacopoeia - however, these methods have not yet been granted compendial status in the United States Pharmacopoeia (USP). In order to facilitate dialogue between stakeholders, the Physicians Committee for Responsible Medicine hosted two virtual roundtable discussions on the perceived barriers to the use of rBET methods for US FDA requirements. Stakeholders agreed that multiple rFC-based methods have been demonstrated to have suitable analytical performance, as described in ICH Q2 on the Validation of Analytical Procedures and USP <1225> on the Validation of Compendial Procedures. United States Pharmacopoeia compendial inclusion of the rFC-based and other rBET methods was favoured, in order to reduce the additional burdens created by a lack of global harmonisation on BET testing requirements.

Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.

An end to horseshoe crab bleeds?

Proposal could allow synthetic proteins to replace harvested enzyme in drug testing.

Endotoxin contamination of nanoparticle formulations: A concern in vaccine adjuvant mechanistic studies.

The increasing awareness of endotoxin contamination has raised important questions during the study of the mechanism of action of the vaccine adjuvants. The endotoxins or lipopolysaccharides (LPS) can contaminate vaccine formulations contributing to result misinterpretations of the in vitro and in vivo studies. In this short communication, we considered the suitability of the Limulus amebocyte lysate (LAL) assay to quantify chitosan (Chit) nanoparticle (NP) endotoxin contamination to use them in a comparative in vitro immunotoxicology study using both LPS-free (LF) and non-LF Chit NPs. It was shown that chit NPs had a masking effect on endotoxin levels, hampering a reliable conclusion about the effect of their contamination. Neither non-LF nor LF Chit NPs induced the production of ROS in RAW 264.7 cells or IL-6 and TNF-α in PBMCs. The lack of effect of non-LF NPs was not expected and likely due to the NPs masking effect, more evident for higher deacetylation degree Chit. Overall, to prevent questionable results, nanomaterials should be produced under endotoxin-free conditions.

Protocol to measure endotoxin from opaque tissues in mice using an optimized kinetic limulus amebocyte lysate assay.

Endotoxin accumulation has been widely noted in several pathologies ranging from metabolic dysregulation to bacterial infection. Using limulus amebocyte lysate (LAL) assays to detect endotoxin load has been the only reliable way to assess endotoxin accumulation, but assays optimized for detection in opaque tissues are still lacking. We optimized a sensitive Kinetic LAL assay for endotoxin detection from murine tissues. In this protocol, we describe tissue collection and homogenization, followed by the procedure to run the assay and data analysis. For complete details on the use and execution of this protocol, please refer to Ceasrine et al. (2022).

The monocyte activation test detects potentiated cytokine release resulting from the synergistic effect of endotoxin and non-endotoxin pyrogens.

Pyrogens are classified in two groups, endotoxin pyrogens and non-endotoxin pyrogens (NEPs). The presence of either in parenteral pharmaceuticals or medical devices can cause severe harm to subjects, and when occurring in combination, synergistic potentiation effects can occur. As the standard in vitro pyrogen test, the Limulus Amebocyte Lysate (LAL) assay can detect LPS only, an endotoxin, but not NEPs. We tested whether the Monocyte Activation Test (MAT) that measures IL-6 induction, is suited for detecting synergistic pyrogen effects. Here we show that MAT reliably detects the NEPs heat-killed Staphylococcus aureus, R848 and lipoteichoic acid, in addition to LPS. When combinations of these pyrogens were tested, a potentiation of IL-6 production was seen beyond an additive effect, apparently reflecting on in-vivo synergisms. The current study therefore demonstrates that MAT not only is a reliable and reproducible assay for the sensitive detection of both endotoxin and non-endotoxin pyrogens, but also for identifying synergistic effects when parenteral drugs are contaminated with multiple pyrogens.

Low endotoxin recovery and its impact on endotoxin detection.

Endotoxin exists on the outer membrane of Gram-negative bacteria and poses risks to human health by triggering a series of immune responses. Therefore, its accurate detection is essential. The Limulus amoebocyte lysate (LAL) test is the most pharmacopeia-recognized and popular technique for endotoxin detection. Despite its wide industry adoption, the low endotoxin recovery (LER) phenomenon can compromise the LAL test's reliability. This review summarizes the possible reasons attributing to the LER phenomenon from three different perspectives: the endotoxin standards used in hold time study, protein active pharmaceutical ingredients, and excipients. Potential mechanisms and strategies to mitigate the LER phenomenon are also discussed as presented by different research groups.

Miniaturization, Parallelization, and Automation of Endotoxin Detection by Centrifugal Microfluidics.

We demonstrate microfluidic automation and parallelization of Limulus amebocyte lysate (LAL)-based bacterial endotoxin testing using centrifugal microfluidics. LAL is the standard reagent to test for endotoxin contaminations in injectable pharmaceuticals. The main features of the introduced system are more than 90% reduction of LAL consumption, from 100 µL/reaction to 9.6 µL/reaction, automated liquid handling to reduce opportunities for contamination and manual handling errors, and microfluidic parallelization by integrating 104 reactions into a single centrifugal microplate. In a single Eclipse microplate, 21 samples and their positive product controls are tested in duplicate. In addition, a standard curve with up to five points is generated, resulting in a total of 104 reactions. Test samples with a defined concentration of 0.5 endotoxin units per milliliter were tested, resulting in a coefficient of variation below 0.75%. A key feature for achieving a small coefficient of variation is ensuring the same path length along the microfluidic channels to the final reaction chambers for each sample and the reagent, so that any unspecific adsorption to the polymer surfaces does not affect the accuracy and precision. Analysis of a sample containing naturally occurring endotoxin with the developed microfluidic microplate yielded comparable results to the conventional testing method. A test with eight commercially available pharmaceuticals was found to pass all requirements for bacterial endotoxin testing as specified in the United States Pharmacopeia. The automated endotoxin testing system reveals specific advantages of centrifugal microfluidics for analytical biochemistry applications. Small liquid volumes are handled (metered, mixed, and aliquoted) in a very precise, highly integrated, and highly parallel manner within mass-fabricated microplates.

Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon.

Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon.

An in vitro study on factors affecting endotoxin neutralization in human plasma using the Limulus amebocyte lysate test.

Endotoxin neutralization, caused by plasma components, makes it difficult to detect endotoxins in human blood. In this study, we investigated which factors influence the recovery of endotoxins using limulus ameobocyte lysate (LAL)-based assays. The individual factors that were examined in more detail were lipoprotein content, type of blood anticoagulation, kinetics and serum levels of divalent cations. Furthermore, it was investigated whether there is a direct correlation between LAL activity and monocyte activation. We could show that polyanionic heparin increases endotoxin recovery in blood, while citrate anticoagulation promotes endotoxin neutralization. Furthermore, we could show that the endotoxin activity in human plasma and serum decreases strongly over time. Time-dependent endotoxin neutralization reaches its maximum after 4-6 h incubation. By means of filtration tests we could determine that endotoxins in the plasma bind to lipoproteins but do not influence their activity. Comparative measurements have shown that high LAL activity of endotoxins in plasma simultaneously possesses high monocyte activating properties in whole blood. For the maximum recovery of endotoxins in human blood the physiological calcium and magnesium concentrations are sufficient. In this study, it was shown that the endotoxin neutralizing plasma components have a molecular weight similar to ß2-microglobulin (11.7 kDa). For the exact identification of the endotoxin neutralizing plasma components, which caused a modulation of the immunostimulating endotoxin activity, further investigations have to be carried out in the future.

Evaluation of limulus amebocyte lysate and recombinant endotoxin alternative assays for an assessment of endotoxin detection specificity.

The United States Pharmacopeia (USP), European Pharmacopeia (EP), and Parenteral Drug Association (PDA) provide guidance on the validation of alternative microbiological methods (U.S. Pharmacopeia National Formulary 2019, Parenteral Drug Association Technical Report No. 33 2013, European Pharmacopoeia (Ph. Eur.) 2017). They define "specificity" as the ability to detect a range of microorganisms. In the context of alternative methods to the compendial Bacterial Endotoxin Test (BET) a range of endotoxins must be considered. This range should represent environmental endotoxins that present risks to pharmaceutical manufacturing processes, final products, and to the most important stakeholder: the patient. This study examines several alternative methods for the bacterial endotoxin detection test. It compares the official and harmonized BET test from two Limulus Amebocyte Lysate (LAL) suppliers to three commercially available recombinant Factor C (rFC) reagents that contain only one of the three enzymes in the horseshoe crab clotting cascade. The study also includes a recombinant reagent that has been developed to include all three of the enzymes involved in the LAL coagulation cascade, occurring in the presence of endotoxins. Pharmaceutically relevant water samples from various points in pharmaceutical water purification processes were used as a source of natural environmental endotoxins. While these water samples are not routinely tested for bacterial endotoxins, they do exist within manufacturing facilities and thus present risks to manufacturing operations (Sandle, 2019). A statistical analysis of 128 samples containing environmental endotoxin has shown that at the 5% level of significance, non-inferiority between the two compendial LAL methods was achieved. However, the non-inferiority claim could not be made with any of the recombinant reagents. The link between the BET and recombinant alternatives remains unresolved and, therefore, requires caution, continued development, and testing.

Establishing a stable platform for the measurement of blood endotoxin levels in the dialysis population.

BACKGROUND: Gram-negative lipopolysaccharides are potent inducers of inflammation and have been shown to be present in patients with end-stage kidney disease. There are a variety of different manufacturers and assay types to quantify endotoxin levels; however, there is no standard methodology to demonstrate its presence in plasma. METHODS: A control group consisting of haemodialysis and non-kidney disease was selected. Five sets of experiments were conducted to try and ascertain the best platform for plasma endotoxin testing. This included: testing of blank tubes; the effects of freezing, thawing and storage on recovery; the effect of different buffers; use of an endpoint assay and comparison of turbidimetric vs. chromogenic kinetic assays. RESULTS: No endotoxin was detected in the blood collection tubes. Freezing and thawing per se did not affect spike recovery rates. However, the sequencing of sample dilution relative to freezing had a significant effect on endotoxin recovery. Buffers increased spike recovery at all levels of dilution. No endotoxin was demonstrated with either the turbidimetric or chromogenic kinetic assay at two different dilutions in the haemodialysis controls. The endpoint assay at a 1:5 dilution did not achieve a valid standard curve. CONCLUSIONS: The findings of our study suggest that, when testing plasma samples, either a turbidimetric or chromogenic assay may be used and should be diluted with appropriate buffers to achieve optimal recovery. Studies looking to quantify the presence of plasma endotoxin need to internally validate their assays and specify their validation findings in their results.

Endotoxin Contamination and Reaction Interfering Substances in the Plant Extract Library.

Endotoxin is an unintentional contaminant that has numerous activities and can affect various biological experiments using cells. In this study, we measured the endotoxin activity of samples from a plant extract library (PEL) and determined their degrees of contamination. Endotoxin was detected in approx. 48% (n = 139) and approx. 4% (n = 5) of field-collected and crude drug samples, respectively, and in concentrations >5.0 EU/mL in some samples. The concentrations of endotoxin that affect cells in vitro vary depending on the target cell type. Although the degree of contamination varied in the present study, it was considered to have little effect on the cell experiments. More than 150 PEL samples had problems with reaction courses or recovery rates of Limulus amoebocyte lysate (LAL) tests. In the LAL tests, using three plant extracts [Sanguisorba officinalis L. (Rosaceae), Oenothera biennis L. (Onagraceae), and Lythrum salicaria L. (Lythraceae)], the polyphenolic compounds in the plant extracts affected LAL test and their effects differed depending on the plant species. When the 16 single polyphenol compounds were added to the LAL tests, the compounds with caffeoyl and pyrogallol moieties were found to affect the LAL reaction course and recovery rate. Furthermore, none of the compounds had any effects at concentrations of 1 µM. Because the plant extracts contained analogs of various polyphenolic compounds, they were presumed to actually act synergistically. Our findings demonstrated that attention must be paid to the recovery rate and reaction process of LAL tests with samples containing polyphenolic compounds.

A novel enzyme immunoassay for the measurement of plasma (1 → 3)-ß-D-glucan levels.

The presence of (1 â†’ 3)-ß-D-glucan in human plasma is a marker for fungal infections. Currently, the Limulus amebocyte lysate (LAL)-based assay is widely used for the quantification of plasma (1 â†’ 3)-ß-D-glucan. However, it has limitations in clinical use, such as an unstable supply of natural resources, complicated manufacturing process, and low-throughput of the reagents. Alternative assays exploiting specific antibodies against (1 â†’ 3)-ß-D-glucan have been developed to overcome these challenges. However, these methods are associated with low sensitivity and poorly correlate with the data obtained by the LAL-based assay. The aim of this study is to develop a novel enzyme immunoassay that is as sensitive and accurate in determining plasma (1 â†’ 3)-ß-D-glucan levels as compared to that obtained with the LAL-based assay. We generated specific monoclonal antibodies against (1 â†’ 3)-ß-D-glucan that recognizes four-unit glucose oligomers with (1 â†’ 3)-ß-D-linkages, and constructed a sandwich enzyme-linked immunosorbent assay (ELISA) using these antibodies. The newly developed ELISA showed proportional increase in absorbance with the volume of (1 â†’ 3)-ß-D-glucan added. The limit of detection of the assay was 4 pg/ml of plasma (1 â†’ 3)-ß-D-glucan that was equivalent to the LAL-based assay and the working range was 4-500 pg/ml. The intra-assay coefficient of variation was 2.2-5.4% using three different concentrations of plasma samples. We observed strong correlation (R = 0.941, slope = 0.986) between the measurements obtained by our ELISA and Fungitec G test ES Nissui, a commonly used LAL-based assay, using 26 types of plasma samples. This could be attributed to the epitopes of the antibodies. Both antibodies could inhibit the LAL-based assay, suggesting that the antibodies recognize the identical regions in ß-D-glucan, thereby inactivating factor G, an initiation zymogen for coagulation cascade, in the LAL-based assay. Thus, the ELISA developed in this study can detect fungal infections in clinical settings with similar efficiency as the LAL-based assay. This assay is characterized by good performance, stable supply of materials, and simple manufacturing process and is more suitable for the high-throughput diagnosis of fungal infections.

Antimicrobial Peptides with Enhanced Salt Resistance and Antiendotoxin Properties.

A strategy was described to design antimicrobial peptides (AMPs) with enhanced salt resistance and antiendotoxin activities by linking two helical AMPs with the Ala-Gly-Pro (AGP) hinge. Among the designed peptides, KR12AGPWR6 demonstrated the best antimicrobial activities even in high salt conditions (NaCl ~300 mM) and possessed the strongest antiendotoxin activities. These activities may be related to hydrophobicity, membrane-permeability, and α-helical content of the peptide. Amino acids of the C-terminal helices were found to affect the peptide-induced permeabilization of LUVs, the α-helicity of the designed peptides under various LUVs, and the LPS aggregation and size alternation. A possible model was proposed to explain the mechanism of LPS neutralization by the designed peptides. These findings could provide a new approach for designing AMPs with enhanced salt resistance and antiendotoxin activities for potential therapeutic applications.

Comparison of bacterial endotoxin testing methods in purified pharmaceutical water matrices.

Current bacterial endotoxin testing systems can be labor-intensive and time-consuming, involving several manual pipetting steps. In our quality control laboratory, annually, we test about 15,000 samples of different grades of purified water, WFI and water samples taken to validate cleaning procedures for endotoxins. We are currently using the Kinetic-QCL™ assay which is a pharmacopeia method that provides reliable results. We compared this assay with another Limulus amebocyte lysate (LAL)-based assay (Endosafe®-MCS) and an alternative endpoint fluorescent recombinant Factor C (rFC) assay (ENDOZYME II GO®). Both these assays have been developed to reduce analyst preparation time. Our objective was to assess if they could increase the throughput of our testing while maintaining low rates of invalid results. The results demonstrated that the two most appropriate methods for rapid endotoxin detection in water are our current assay, K-QCL, and the rFC-based assay, ENDOZYME II GO. This latter assay was found to be less sensitive to interference than our current assay, particularly in cleaning validation water samples. It also showed better performance, accuracy, repeatability and had a shorter time-to-results. ENDOZYME II GO assay allows quick testing of large numbers of samples with reliable results and is a good alternative for conventional LAL assays.

Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review.

Endotoxin testing by recombinant factor C (rFC) is increasing with the addition of new suppliers of reagents. By use of a recombinantly produced factor C , based on the sequence of a coagulation enzyme present in horseshoe crab amebocyte lysates, the rFC tests are designed as substitutes for the traditional Limulus amebocyte lysate (LAL)/Tachypleus amebocyte lysate tests based on horseshoe crab blood. Comparative testing of samples with both the LAL and recombinant reagents has shown a high degree of correlation, suggesting that use of rFC is comparable to the more traditional LAL tests and may be technologically superior. Recombinant factor C does not recognize the factor G pathway, the alternate coagulation pathway that the lysate reagents detect. This feature allows rFC to detect endotoxin more selectively. As a recombinantly produced material, it avoids the use of the horseshoe crabs required for lysate production, thereby protecting this species, which is at risk in some parts of the world. Recombinant factor C is expected to further benefit from a more sustainable supply chain based upon a robust biotechnological production process. We summarize here the results of many studies that evaluated the use of recombinant technology for the detection of environmental endotoxin. Additionally, we include a review of the current compendia and regulatory status of the recombinant technologies for use in the quality control of pharmaceutical manufacturing. Our analysis confirms that the recombinant technologies are comparable in protecting patient safety.