Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays
Summary
Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.
Abstract
When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.
Introduction
An endotoxin is a building block of the Gram-negative bacterial cell wall1,2. It can activate the immune cells at very low (picogram) concentrations1,2. The proinflammatory mediators (cytokines, leukotrienes, eicosanoids, etc.) produced by the cells in response to an endotoxin are responsible for fever, hypotension, hypertension, and more severe health problems including multiple organ failure1,2,3. The severity of the immune-mediated side-effects triggered by the endotoxin depends on its potency determined by the endotoxin composition and structure and measured in international endotoxin units (IUs or EUs)3. The number of these units per kilogram of body weight is used to set a threshold pyrogenic dose of endotoxin. This dose is 5 EU/kg for drug products administered via all routes but the intrathecal route. Drugs dosed per square meter of body surface, intraocular fluids, radiopharmaceuticals, and products administered via intrathecal route have a different threshold pyrogenic dose, which is 100 EU/m2, 0.2 EU/mL, 175 EU/V (where V is the volume of the product intended for administration), and 0.2 EU/kg, respectively4. More details about the threshold pyrogenic dose for various drug products and devices are provided and discussed elsewhere4,5,6.
Animals vary widely in their sensitivity to endotoxin-mediated reactions. Humans, non-human primates, and rabbits are among the species most extremely sensitive to endotoxins3. To avoid endotoxin-mediated side effects in patients and prevent inaccurate conclusions of preclinical toxicity and efficacy studies, it is essential to accurately detect and quantify endotoxins in both clinical and pre-clinical grade formulations. Several currently available methods can achieve this task. One of them is the Limulus Amoebocyte Lysate (LAL) assay, which is commonly used worldwide to screen biomedical products for the potential endotoxin contamination as well as to detect bacterial infections7,8,9. The lysate is prepared from amoebocytes, the cells present in the blood of horseshoe crabs Limulus polyphemus residing in the east shore of the continent of North America7. Interestingly, there are a few different species of horseshoe crabs (Tachypleus gigas and Tachypleus tridentatus) in Asia10. The Tachypleus Amoebocyte Lysate (TAL) is used in several Asian countries for the detection of endotoxin similar to how the LAL is used in other cuntries10. The lysates (LAL and TAL) contain a group of proteins that upon activation confer protease activity. One of these proteins, the so-called Factor C is activated upon contact with endotoxin. Activated Factor C cleaves Factor B, which in turn also becomes a protease and cleaves a pro-clotting enzyme to produce a clotting enzyme. The result of this chain of reactions is the formation of a gel, an increase in the sample turbidity and, in the presence of a chromogenic substrate, the appearance of a colored product, which serve as a foundation for gel-clot, turbidity, and chromogenic assays, respectively. While there is no mandatory LAL format, the US Food and Drug Administration (FDA) explains in the guidance for industry document, that in case of discrepancy in the test results between different LAL formats, the decision is made based on the gel-clot assay5.
Many commonly used laboratory chemicals (e.g., EDTA) and known drug products (e.g., penicillin) interfere with LAL assays11. The interference is usually identified by assessing the recovery of the endotoxin standard spiked at a known concentration into a solution containing the test material. If the spike recovery is less than 50% or more than 200%, then the result of the LAL assay for the given test material is invalid due to the inhibition or enhancement, respectively4. Nanotechnology-based formulations are often complex and interfere with the LAL through a variety of mechanisms12,13,14. Many approaches have been described to overcome the interference: sample reconstitution in specific buffers and surfactants, protein inactivation by heating, destruction of lipid-based hollow materials by heating and supplementing the sample with excess divalent cations5,12,13,14,15. Alternative methods for situations when LAL interference cannot be overcome have also been described: ELISA, a HEK-TLR4 reporter cell line assay, and mass spectrometry16,17,18,19.
Herein, experimental procedures for conducting gel-clot, turbidity, and chromogenic LAL assays are described. These assays are also available on the Nanotechnology Characterization Lab (NCL) website20 in protocols STE1.2 (turbidity LAL), STE1.3 (gel-clot LAL) and STE1.4 (chromogenic LAL). It is recommended to conduct at least two different formats to characterize the same nano-formulation. When results of the turbidity and chromogenic LAL disagree, the gel-clot results are considered5. When results of two LAL formats disagree, additional studies using either monocyte activation test (MAT) or rabbit pyrogen test (RPT) to verify LAL findings are conducted21. It is important to note that each method used for endotoxin detection and pyrogenicity assessment has advantages and limitations21,22,23,24. Recognizing limitations of the procedure used to characterize a given nanotechnology formulation is essential to obtain scientific justification for the use of the procedure optimal for that nano-formulation.
In this study, PEGylated liposomal doxorubicin was used as a model nanoparticle formulation. This formulation has been approved by the US FDA in 1995 and used for treating cancer patients worldwide25.
Protocol
Representative Results
Discussion
The information provided in this protocol has been described before15,26 and relies on several regulatory documents published by the US Food and Drug Administration (US FDA or FDA) and United States Pharmacopoeia (USP)4,5,6,27, and is also available on the NCL website20 in protocols STE1.2 (turbidity LAL), STE1.3…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The study was supported by federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
Materials
| Turbidity LAL Assay | |||
| Sodium Hydroxide | Sigma | S2770 | When needed, it is used to adjust sample pH to be between 6-8 |
| Hydrochloric acid | Sigma | H9892 | When needed, it is used to adjust sample pH to be between 6-8 |
| LAL Reagent | Associates of Cape Cod | T0051 | This reagent can be used with turbidity assay only |
| Control Endotoxin Standard | Associates of Cape Cod | E0005 | This reagent can be used with turbidity and gel-clot assays |
| LAL grade water | Associates of Cape Cod | WP0501 | This reagent can be used with any LAL format |
| Glucashield Buffer | Associates of Cape Cod | GB051-25 | Used to prevent false-positive response from beta-glucans |
| Disposable endotoxin-free glass dilution tubes 12 x 75 mm | Associates of Cape Cod | TB240 | These tubes can be used with all three assays |
| Disposable endotoxin-free glass reaction tubes 8 x 75 mm | Associates of Cape Cod | TK100 | These tubes can be used with turbidity and chromogenic assays |
| Pyrogen-free tips with volumes 0.25 and 1.0 mL | RAININ | PPT25, PPT10 | Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed |
| Pyrogen-free microcentrifuge tubes, 2.0 mL | Eppendorf | 22600044 | Other equivalent supplies can be used |
| Pyrogen-fee combitips, 5mL | Eppendorf | 30089669 | Other equivalent supplies can be used |
| Repeat pipettor | Eppendorf | 4982000020 | Other equivalent supplies can be used |
| Microcetrifuge | any brand | Any brand can be used | |
| Refrigerator, 2-8 C | any brand | Any brand can be used | |
| Vortex | any brand | Any brand can be used | |
| Freezer, -20 C | any brand | Any brand can be used | |
| Pyros Kinetix or Pyros Kinetix Flex reader | Associates of Cape Cod | PKF96 | Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions |
| Chromogenic LAL Assay | |||
| Pyrochrome LAL Reagent | Associates of Cape Cod | CG1500-5 | This reagent is specific to the Chromogenic Assay |
| Control Endotoxin Standard | Associates of Cape Cod | EC010 | This standard is different than that used for turbidity and gel-clot LALs; it is optimized for optimal performance in the chromogenic assay |
| Sodium Hydroxide | Sigma | S2770 | When needed, it is used to adjust sample pH to be between 6-8 |
| Hydrochloric acid | Sigma | H9892 | When needed, it is used to adjust sample pH to be between 6-8 |
| LAL grade water | Associates of Cape Cod | WP0501 | This reagent can be used with any LAL format |
| Glucashield Buffer | Associates of Cape Cod | GB051-25 | Used to prevent false-positive response from beta-glucans |
| Disposable endotoxin-free glass dilution tubes 12 x 75 mm | Associates of Cape Cod | TB240 | These tubes can be used with all three assays |
| Disposable endotoxin-free glass reaction tubes 8 x 75 mm | Associates of Cape Cod | TK100 | These tubes can be used with turbidity and chromogenic assays |
| Pyrogen-free tips with volumes 0.25 and 1.0 ml | RAININ | PPT25, PPT10 | Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed |
| Pyrogen-free microcentrifuge tubes, 2.0 mL | Eppendorf | 22600044 | Other equivalent supplies can be used |
| Pyrogen-fee combitips, 5mL | Eppendorf | 30089669 | Other equivalent supplies can be used |
| Repeat pipettor | Eppendorf | 4982000020 | Other equivalent supplies can be used |
| Microcetrifuge | any brand | Any brand can be used | |
| Refrigerator, 2-8 C | any brand | Any brand can be used | |
| Vortex | any brand | Any brand can be used | |
| Freezer, -20 C | any brand | Any brand can be used | |
| Pyros Kinetix or Pyros Kinetix Flex reader | Associates of Cape Cod | PKF96 | Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions |
| Gel-Clot LAL Assay | |||
| LAL Reagent | Associates of Cape Cod | G5003 | This reagent is specific to the gel-clot assay |
| Control Endotoxin Standard | Associates of Cape Cod | E0005 | This reagent can be used with turbidity and gel-clot assays |
| Sodium Hydroxide | Sigma | S2770 | When needed, it is used to adjust sample pH to be between 6-8 |
| Hydrochloric acid | Sigma | H9892 | When needed, it is used to adjust sample pH to be between 6-8 |
| LAL grade water | Associates of Cape Cod | WP0501 | This reagent can be used with any LAL format |
| Glucashield Buffer | Associates of Cape Cod | GB051-25 | Used to prevent false-positive response from beta-glucans |
| Disposable endotoxin-free glass dilution tubes 12 x 75 mm | Associates of Cape Cod | TB240 | These tubes can be used with all three assays |
| Disposable endotoxin-free glass reaction tubes 10 x 75 mm | Associates of Cape Cod | TS050 | These tubes are for use with the gel-clot assay |
| Pyrogen-free tips with volumes 0.25 and 1 mL | RAININ | PPT25, PPT10 | Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed |
| Pyrogen-free microcentrifuge tubes, 2.0 mL | Eppendorf | 22600044 | Other equivalent supplies can be used |
| Pyrogen-fee combitips, 5mL | Eppendorf | 30089669 | Other equivalent supplies can be used |
| Repeat pipettor | Eppendorf | 4982000020 | Other equivalent supplies can be used |
| Microcetrifuge | any brand | Any brand can be used | |
| Refrigerator, 2-8 C | any brand | Any brand can be used | |
| Vortex | any brand | Any brand can be used | |
| Freezer, -20 C | any brand | Any brand can be used | |
| Water bath, 37 C | any brand | Any brand can be used, however, it is important either to switch off water circulation or use non-circualting water bath because water flow will affect clot formation and lead to false-negative results |
References
- Perkins, D. J., Patel, M. C., Blanco, J. C., Vogel, S. N. Epigenetic Mechanisms Governing Innate Inflammatory Responses. Journal of Interferon & Cytokine Research. 36 (7), 454-461 (2016).
- Vogel, S. N., Awomoyi, A. A., Rallabhandi, P., Medvedev, A. E. Mutations in TLR4 signaling that lead to increased susceptibility to infection in humans: an overview. Journal of Endotoxin Research. 11 (6), 333-339 (2005).
- Dobrovolskaia, M. A., Vogel, S. N. Toll receptors, CD14, and macrophage activation and deactivation by LPS. Microbes and Infection. 4 (9), 903-914 (2002).
- US Pharmacopeia. . Bacterial Endotoxins Test. , (2011).
- FDA, U. . Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers. , (2012).
- FDA, U. . Endotoxin Testing Recommendations for Single-Use Intraocular Ophthalmic Devices. , (2015).
- Fennrich, S., et al. More than 70 years of pyrogen detection: Current state and future perspectives. Alternatives to Laboratory Animals. 44 (3), 239-253 (2016).
- Kumar, M. S., Ghosh, S., Nayak, S., Das, A. P. Recent advances in biosensor based diagnosis of urinary tract infection. Biosensors and Bioelectronics. 80, 497-510 (2016).
- Solano, G., Gomez, A., Leon, G. Assessing endotoxins in equine-derived snake antivenoms: Comparison of the USP pyrogen test and the Limulus Amoebocyte Lysate assay (LAL). Toxicon. , 13-18 (2015).
- Akbar John, B., Kamaruzzaman, B. Y., Jalal, K. C. A., Zaleha, K. TAL – a source of bacterial endotoxin detector in liquid biological samples. International Food Research Journal. 19 (2), 423-425 (2012).
- Fujita, Y., Tokunaga, T., Kataoka, H. Saline and buffers minimize the action of interfering factors in the bacterial endotoxins test. Analytical Biochemistry. 409 (1), 46-53 (2011).
- Dobrovolskaia, M. A. Pre-clinical immunotoxicity studies of nanotechnology-formulated drugs: Challenges, considerations and strategy. Journal of Controlled Release. 220 (Pt B), 571-583 (2015).
- Dobrovolskaia, M. A., et al. Ambiguities in applying traditional Limulus amebocyte lysate tests to quantify endotoxin in nanoparticle formulations. Nanomedicine (London). 5 (4), 555-562 (2010).
- Dobrovolskaia, M. A., Neun, B. W., Clogston, J. D., Grossman, J. H., McNeil, S. E. Choice of method for endotoxin detection depends on nanoformulation. Nanomedicine (London). 9 (12), 1847-1856 (2014).
- Neun, B. W., Dobrovolskaia, M. A. Considerations and Some Practical Solutions to Overcome Nanoparticle Interference with LAL Assays and to Avoid Endotoxin Contamination in Nanoformulations. Methods in Molecular Biology. 1682, 23-33 (2018).
- Boratynski, J., Szermer-Olearnik, B. Endotoxin Removal from Escherichia coli Bacterial Lysate Using a Biphasic Liquid System. Methods in Molecular Biology. 1600, 107-112 (2017).
- Li, H., Hitchins, V. M., Wickramasekara, S. Rapid detection of bacterial endotoxins in ophthalmic viscosurgical device materials by direct analysis in real time mass spectrometry. Analytica Chimica Acta. 943, 98-105 (2016).
- Uhlig, S., et al. Profiling of 3-hydroxy fatty acids as environmental markers of endotoxin using liquid chromatography coupled to tandem mass spectrometry. Journal of Chromatography A. 1434, 119-126 (2016).
- Smulders, S., et al. Contamination of nanoparticles by endotoxin: evaluation of different test methods. Particle and Fibre Toxicology. 9, 41 (2012).
- . NCL assay cascade Available from: https://ncl.cancer.gov/resources/assay-cascade-protocols (2015)
- Dobrovolskaia, M. A., Germolec, D. R., Weaver, J. L. Evaluation of nanoparticle immunotoxicity. Nature Nanotechnology. 4 (7), 411-414 (2009).
- Borton, L. K., Coleman, K. P. Material-mediated pyrogens in medical devices: Applicability of the in vitro Monocyte Activation Test. Altex. , (2018).
- Stoppelkamp, S., et al. Speeding up pyrogenicity testing: Identification of suitable cell components and readout parameters for an accelerated monocyte activation test (MAT). Drug Testing and Analysis. 9 (2), 260-273 (2017).
- Vipond, C., Findlay, L., Feavers, I., Care, R. Limitations of the rabbit pyrogen test for assessing meningococcal OMV based vaccines. Altex. 33 (1), 47-53 (2016).
- Barenholz, Y. Doxil(R)–the first FDA-approved nano-drug: lessons learned. Journal of Controlled Release. 160 (2), 117-134 (2012).
- Neun, B. W., Dobrovolskaia, M. A. Detection and quantitative evaluation of endotoxin contamination in nanoparticle formulations by LAL-based assays. Methods in Molecular Biology. 697, 121-130 (2011).
- FDA, U. . Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. , (2005).
- Mohan, P., Rapoport, N. Doxorubicin as a molecular nanotheranostic agent: effect of doxorubicin encapsulation in micelles or nanoemulsions on the ultrasound-mediated intracellular delivery and nuclear trafficking. Molecular Pharmaceutics. 7 (6), 1959-1973 (2010).
- Dabbagh, A., et al. Low-melting-point polymeric nanoshells for thermal-triggered drug release under hyperthermia condition. International Journal of Hyperthermia. 31 (8), 920-929 (2015).
- Li, Y., et al. Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles. Nanotoxicology. 9 (4), 462-473 (2015).
- Li, Y., et al. Bacterial endotoxin (lipopolysaccharide) binds to the surface of gold nanoparticles, interferes with biocorona formation and induces human monocyte inflammatory activation. Nanotoxicology. 11 (9-10), 1157-1175 (2017).
