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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 tri....

Protocol

1. Preparation of Nanoparticle Samples

  1. Prepare the study sample in LAL grade water.
  2. If the sample pH is outside of the 6-8 range, adjust the pH by using pyrogen-free sodium hydroxide or hydrochloric acid.
  3. Using LAL grade water prepare several dilutions of the study sample. Make sure that the highest dilution does not exceed maximum valid dilution (MVD). Refer to the discussion section for details about MVD estimation.

2. Preparation of Reagents Common Betwe.......

Representative Results

The example of data generated after testing this formulation in LAL assays is shown in Table 1. PEGylated liposomal doxorubicin interfered with chromogenic LAL at dilution 5. However, this interference was overcome by greater dilutions. Spike recovery was between 50 and 200% when this formulation was tested at dilutions 50 and 500 in turbidity and chromogenic LAL, as well as at dilution 5 in turbidity LAL. When adjusted by the dilution factor, the results were consistent .......

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.......

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

NameCompanyCatalog NumberComments
Turbidity LAL Assay
Sodium HydroxideSigmaS2770When needed, it is used to adjust sample pH to be between 6-8
Hydrochloric acidSigmaH9892When needed, it is used to adjust sample pH to be between 6-8
LAL ReagentAssociates of Cape CodT0051This reagent can be used with turbidity assay only
Control Endotoxin StandardAssociates of Cape CodE0005This reagent can be used with turbidity and gel-clot assays
LAL grade waterAssociates of Cape CodWP0501This reagent can be used with any LAL format
Glucashield BufferAssociates of Cape CodGB051-25Used to prevent false-positive response from beta-glucans
Disposable endotoxin-free glass dilution tubes 12 x 75 mmAssociates of Cape CodTB240These tubes can be used with all three assays
Disposable endotoxin-free glass reaction tubes 8 x 75 mmAssociates of Cape CodTK100These tubes can be used with turbidity and chromogenic assays
Pyrogen-free tips with volumes 0.25 and 1.0 mLRAININPPT25, PPT10Tips 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 mLEppendorf22600044Other equivalent supplies can be used
Pyrogen-fee combitips, 5mLEppendorf30089669Other equivalent supplies can be used
Repeat pipettorEppendorf4982000020Other equivalent supplies can be used
Microcetrifugeany brandAny brand can be used
Refrigerator, 2-8 Cany brandAny brand can be used
Vortexany brandAny brand can be used
Freezer, -20 Cany brandAny brand can be used
Pyros Kinetix or Pyros Kinetix Flex readerAssociates of Cape CodPKF96Other 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 ReagentAssociates of Cape CodCG1500-5This reagent is specific to the Chromogenic Assay
Control Endotoxin StandardAssociates of Cape CodEC010This standard is different than that used for turbidity and gel-clot LALs; it is optimized for optimal performance in the chromogenic assay
Sodium HydroxideSigmaS2770When needed, it is used to adjust sample pH to be between 6-8
Hydrochloric acidSigmaH9892When needed, it is used to adjust sample pH to be between 6-8
LAL grade waterAssociates of Cape CodWP0501This reagent can be used with any LAL format
Glucashield BufferAssociates of Cape CodGB051-25Used to prevent false-positive response from beta-glucans
Disposable endotoxin-free glass dilution tubes 12 x 75 mmAssociates of Cape CodTB240These tubes can be used with all three assays
Disposable endotoxin-free glass reaction tubes 8 x 75 mmAssociates of Cape CodTK100These tubes can be used with turbidity and chromogenic assays
Pyrogen-free tips with volumes 0.25 and 1.0 mlRAININPPT25, PPT10Tips 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 mLEppendorf22600044Other equivalent supplies can be used
Pyrogen-fee combitips, 5mLEppendorf30089669Other equivalent supplies can be used
Repeat pipettorEppendorf4982000020Other equivalent supplies can be used
Microcetrifugeany brandAny brand can be used
Refrigerator, 2-8 Cany brandAny brand can be used
Vortexany brandAny brand can be used
Freezer, -20 Cany brandAny brand can be used
Pyros Kinetix or Pyros Kinetix Flex readerAssociates of Cape CodPKF96Other 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 ReagentAssociates of Cape CodG5003This reagent is specific to the gel-clot assay
Control Endotoxin StandardAssociates of Cape CodE0005This reagent can be used with turbidity and gel-clot assays
Sodium HydroxideSigmaS2770When needed, it is used to adjust sample pH to be between 6-8
Hydrochloric acidSigmaH9892When needed, it is used to adjust sample pH to be between 6-8
LAL grade waterAssociates of Cape CodWP0501This reagent can be used with any LAL format
Glucashield BufferAssociates of Cape CodGB051-25Used to prevent false-positive response from beta-glucans
Disposable endotoxin-free glass dilution tubes 12 x 75 mmAssociates of Cape CodTB240These tubes can be used with all three assays
Disposable endotoxin-free glass reaction tubes 10 x 75 mmAssociates of Cape CodTS050These tubes are for use with the gel-clot assay
Pyrogen-free tips with volumes 0.25 and 1 mLRAININPPT25, PPT10Tips 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 mLEppendorf22600044Other equivalent supplies can be used
Pyrogen-fee combitips, 5mLEppendorf30089669Other equivalent supplies can be used
Repeat pipettorEppendorf4982000020Other equivalent supplies can be used
Microcetrifugeany brandAny brand can be used
Refrigerator, 2-8 Cany brandAny brand can be used
Vortexany brandAny brand can be used
Freezer, -20 Cany brandAny brand can be used
Water bath, 37 Cany brandAny 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

  1. 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).
  2. Vogel, S. N., Awomoyi, A. A., Rallabhandi, P., Medvedev, A. E.

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