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 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 elsewhere^4,5,6.

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 infections^7,8,9. 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 mechanisms^12,13,14. 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 cations^{5,12,13,14,15}. Alternative methods for situations when LAL interference cannot be overcome have also been described: ELISA, a HEK-TLR4 reporter cell line assay, and mass spectrometry^16,17,18,19.

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 limitations^21,22,23,24. 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

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 concentration of 0.1 N.
2. Dilute concentrated hydrochloric acid stock using pyrogen-free LAL reagent water and prepare a working solution at a final concentration of 0.1 N.
3. Preparation of the Control Standard Endotoxin (CSE)
   1. Reconstitute the CSE according to the certificate of analysis supplied by the manufacturer.
      NOTE: Refer to the discussion section for important notes regarding the information provided in the certificate. Refer to the Table of Materials for the details regarding catalog number and application of a given CSE formulation in different LAL formats.
4. Preparation of the LAL Reagent
   1. Reconstitute the LAL reagent according to the certificate of analysis provided by the manufacturer.
      NOTE: Refer to the discussion section for important details regarding LAL reagent preparation. Refer to the Table of Materials for the details regarding catalog number and application of a given LAL reagent formulation in different LAL format.

3. Turbidity LAL Assay

1. Preparation of the Calibration Standards
   1. Using 900 µL of LAL grade water and 100 µL of CSE, prepare as many intermediate dilutions as needed to enable the preparation of a calibration standard with a concentration range from 0.001 to 1 EU/mL.
   2. First label tubes and add 900 μL of LAL-grade water into each tube. Then add 100 μL of 10 EU/mL solutionn to prepare calibration standard with concentration of 1EU/mL.
   3. Repeat the serial 10-fold dilution as described above to prepare three lower calibration standards. Verify that four calibration standards ranging from 0.001 to 1 EU/mL have been prepared.
2. Preparation of the Quality Controls
   1. Prepare a 0.05 EU/mL quality control by combining 50 µL of the 1 EU/mL CSE solution with 950 µL of LAL-grade water.
      NOTE: Refer to the discussion section for details regarding control preparation.
3. Preparation of Inhibition/Enhancement (IEC) Controls
   1. Prepare IEC with concentration of 0.05 EU/mL by combining 25 µL of the 1 EU/mL CSE solution and 475 µL of the test nanomaterial at a given dilution.
      NOTE: Refer to the discussion section for additional details.
4. Experimental procedure
   1. Allow the instrument to warm up by turning it on approximately 30 min in advance. Set-up the detection wavelength to 660 nm as this is appropriate for the turbidity LAL.
   2. Sign in by typing the username and password.
   3. Open the software (Table of Materials) by clicking on the corresponding icon on a computer screen.
   4. Select Collect data on the software home screen. Enter the test ID and data group information into the corresponding space in the General tab on the home screen.
   5. Click the Hardware tab. Choose the instrument type from a dropdown menu.
   6. Set-up the detection wavelength to 660 nm as this is appropriate for the turbidity LAL by chosing the LAL turbidity method.
   7. Verify that a serial number, system ID and serial port information appear on the screen. Click OK. Click OK one more time to confirm.
   8. Enter the sample ID in the same order the sample is tested. Use default buttons to enter the negative control, standard curve and test samples. 
   9. Prepare duplicate tubes for each sample and add 200 µL (test ratio 4:1) or 100 µL (test ratio 1:1) of negative control (water), calibration standards, quality control, IEC and test nanoparticles into pre-labeled glass tubes.
   10. Add 50 µL (test ratio 4:1) or 100 µL (test ratio 1:1) of LAL reagent to first test vial, vortex it briefly, and insert into test slot in the instrument carousel. If the 1:1 ratio is used, the volume of the LAL reagent is 100 µL.
   11. Repeat the procedure described above for other samples. Process samples one at a time.
       NOTE: Refer to the discussion section for more details.

4. Chromogenic LAL

1. Preparation of Calibration Standards
   1. Using 900 µL of LAL grade water and 100 µL of CSE, prepare as many intermediate dilutions as needed to enable the preparation of a calibration standard with a concentration of 1 EU/mL.
   2. Using 900 µL LAL-grade water and 100 µL of the 1 EU/mL calibration standard, prepare a second calibration standard at a concentration of 0.1 EU/mL.
   3. Repeat the serial 10-fold dilution as described above to prepare two lower calibration standards. Verify that four calibration standards ranging from 0.001 to 1 EU/mL have been prepared.
2. Preparation of Quality Controls.
   1. Prepare a 0.05 EU/mL quality control by combining 50 µL of the 1 EU/mL CSE solution with 950 µL of LAL-grade water.
      NOTE: Refer to the discussion section for details regarding control preparation.
3. Preparation of Inhibition/Enhancement (IEC) Controls
   1. Prepare 0.05 EU/mL by combining 25 μL of the 1 EU/mL CSE solution with 475 μL of test nanomaterial.
      NOTE: Refer to the discussion section for additional details.
4. Experimental procedure
   1. Allow the instrument to warm up by turning it on approximately 30 min in advance. Set-up the detection wavelength to 405 nm as this is appropriate for the turbidity LAL.
   2. Open the software by clicking on the corresponding icon on a computer screen. Sign in by typing the username and password.
   3. Select Collect data on the software home screen. Enter test ID and data group information into corresponding space in the General tab on the home screen.
   4. Click the Hardware tab. Choose the instrument type from a dropdown menu. Choose the instrument.
   5. Verify that a serial number, system ID and serial port information appear on the screen. Click OK. Click OK one more time to confirm.
   6. Enter the sample ID in the same order the sample is tested. Use default buttons to enter negative control, standard curve and test samples.
   7. Prepare duplicate tubes for each sample and add 200 µL (test ratio 4:1) or 100 µL (test ratio 1:1) of negative control (water), calibration standards, quality control, IEC and test nanoparticles into pre-labeled glass tubes.
   8. Add 50 µL (test ratio 4:1) or 100 µL (test ratio 1:1) of LAL reagent to first test vial, vortex it briefly, and insert into test slot in the instrument carousel. If the 1:1 ratio is used, the volume of the LAL reagent is 100 µL.
   9. Repeat the procedure described above for other samples. Process samples one at a time.

5. Gel-Clot LAL

NOTE: This assay identifies the presence of endotoxins in the sample based on the visual observation and detection of a clot in the reaction tube. The experimental steps are described below. Use a bench sheet to record the results. This bench sheet is not mandatory, and other ways of recording the assay results are also acceptable. An example of such a bench sheet is provided in supplementary materials for the convenience of a reader. Lambda (l) is the sensitivity of the gel-clot assay and is 0.03 EU/mL.

1. Label as many reaction tubes as needed to accommodate the number of analyzed test samples. Refer to the bench sheet for details about the number of replicates used in step 1, step 2 and step 3 of the assay.
2. Aliquot 100 μL of water, controls or test sample per tube.
3. Prepare CSE such that the final concentration is equal to 4λ.
4. Combine 100 μL of the standard mentioned above with 100 μL of water or test sample to achieve the final concentration of CSE of 2λ. Repeat three more times to achieve lambda and half-lambda and a quarter lambda.
5. Make sure that the temperature in the water bath is 37 °C.
6. Add 100 μL of lysate per test tube, vortex briefly and place the rack with all the tubes into the water bath for 1 h.
7. Invert the tube with a smooth motion.
8. Manually record results using “+” (firm clot) or “-“ (no clot or loose clot) on the bench sheet.
9. Proceed with the analysis according to the USP BET 85⁴;use the bench sheet as supporting material

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

List of materials used in this article

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