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 wall^1,2. It can activate the immune cells at very low (picogram) concentrations^1,2. 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 failure^1,2,3. 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 Between LAL Formats

1. Dilute concentrated sodium hydroxide stock using pyrogen-free LAL reagent water to prepare a working solution at a concen....

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 before^15,26 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.......

Materials

Name	Company	Catalog Number	Comments
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