Bacterial Endotoxin Testing, Part 2: Prerequisites & Reagent Preparation

By Yadnyesh Patel, microbiology subject matter expert

All injectable pharmaceutical products and implantable medical devices that come into contact with the bloodstream or spinal fluid are tested for endotoxins using bacterial endotoxin testing (BET) to ensure that the sterile products are safe for human use. In my last article, I provided an overview of BET. In this second article of a planned series, I share best practices about prerequisites and reagent preparation for a gel clot Limulus amebocyte lysate (LAL) test.

Preparatory Requirements

Bacterial endotoxin testing (BET) can be performed in most modern laboratories under controlled conditions. Appropriate aseptic technique is important when preparing and diluting standards and handling samples. Gowning practice outside of normal laboratory personal protective equipment (PPE) requirements is not a concern unless the product under test demands specific analyst safety considerations due to toxicity or infectiousness. Gloves should be talc-free, as the talc may contain significant levels of endotoxins. Plate readers, water baths, and dry heat blocks used for sample incubation should be on a laboratory bench away from heating, ventilation, and air conditioning (HVAC) ducts, significant vibration, and laboratory traffic that could affect the test results. Sample hold times and conditions should be determined and subsequently documented, if necessary, to ensure that accurate test results can be generated in the qualified time. For example, if the laboratory receives a water for injection (WFI) or in-process sample, must it be refrigerated or can it remain at room temperature, and for how long? Prior to testing, it is recommended that the primary sample container(s) be adequately mixed before removing the test aliquot(s) for either direct testing or subsequent dilution.

Reference Standard Endotoxin (RSE) And Control Standard Endotoxin (CSE) Calibration

It is well known that purified lipopolysaccharides (LPS) from different genera/species/strains of Gram-negative bacteria (GNB), when experimentally administered to rabbits on a uniform weight or mass basis, can produce significantly different pyrogenic reactions. However, by relating these effects to the administered activity, meaning their ability to elicit a fever in the rabbit or initiate an LAL reaction rather than weight or mass, the structural variability of LPS molecules can be normalized to a defined unit of activity called the endotoxin unit (EU).

Two notes apply here: 


• One USP EU is equivalent to one international unit (IU) as indicated in USP <85>.
• Lysate reagents currently described in USP <85> are licensed in the United States by the FDA and have a sensitivity associated 
with them; however, the FDA does not license CSE preparations.


The RSE is the purified LPS primary standard that is formulated from a common bulk preparation of E. coli 0113:H10:K LPS
that is shared among the World Health Organization (WHO), European Pharmacopoeia (Ph.Eur.), Japanese Pharmacopoeia (JP), and U.S. Pharmacopeia (USP). The RSE was originally developed by the FDA in the 1970s to calibrate lysate reagents from multiple manufacturers, and it remains the primary standard for lysate calibration, calibration of secondary standards (e.g., CSE), and the generation of assay parameters such as standard curves and PPC assay controls. 
Although RSE is available from USP for routine use, most BET assays are performed using a CSE, which is a secondary calibration analyte that may be included in LAL test kits purchased from reagent manufacturers. Many CSEs are provided as lyophilized preparations of a purified LPS that was filled and packaged by weight, not by activity. The purpose of the standardization study is to determine the specific activity, or potency, of the CSE in EU/unit of weight of the material against the RSE primary standard. 
All the reagents used in BET assays are biological in nature and, therefore, can exhibit some variability in sensitivity and potency, making calibration against the RSE an important task. Calibration against RSE is necessary for each unique combination of lysate lot and CSE lot. If a kit is purchased from a reagent vendor, the vendor will conduct the lot-specific standardization and provide a lot-specific certificate of analysis (COA) in the kit, which should be retained for reference in the laboratory. However, there may be circumstances when a laboratory might choose to purchase CSE from a third party or to calibrate a liquid endotoxin preparation, which requires that the laboratory conduct its own calibration study 


CSE Calibration/Potency Determination Using the Gel Clot Method

CSE potency determination in the gel clot test is accomplished by comparing the geometric mean (GM) endpoints of separate dilution series of RSE and CSE made in Water for BET and tested in quadruplicate. The endpoint is defined as the last tube in a two-fold series of RSE or CSE dilutions showing a positive reaction (gel). To have an endpoint, the last positive tube must be followed in the series by at least one negative dilution.

The GM is calculated as follows:

Geometric mean = Antilog (Σlog₁₀endpoints / Number of replicates)

RSE is labeled in units of activity (EU/vial), and the units for the GM endpoint for the RSE are expressed as EU/mL. Because the CSE is filled by weight, the units for the GM endpoint for the CSE are expressed as ng/mL. The potency of the CSE in EU/ng is calculated as follows:

                                                                            GM endpoint of the RSE series in EU/mL

Potency of CSE (EU/ng) =        ------------------------------------------------------------

                                                                            GM endpoint of the CSE series in ng/mL

For example, to calibrate a new lot of CSE for use with a gel-clot lysate lot with a labeled sensitivity of 0.125 EU/mL, dilute the RSE in EU/mL to bracket the label claim of the lysate reagent. For this example, the GM of the RSE confirms the label claim of 0.125 EU/mL. Dilute the CSE in ng/mL so that an endpoint will be reached. For this example, the endpoint of the CSE series is 0.00625 ng/mL. The potency of the CSE for the particular lysate lot is calculated as follows:

GM of the RSE =     0.125 EU/mL      =  20 EU/ng of CSE
GM of the CSE         0.00625 ng/mL

For this lot of lysate, the potency of the new CSE lot is 20 EU/ng. If the lab wants to use this CSE lot with a different lysate lot, the calibration study must be repeated because potency determination references only a specific combination of lysate lot and CSE lot.

Screening and Qualification of Consumables

If employing plastic apparatus, such as microplates and pipet tips for automatic pipettes, use apparatus that is shown to be free of detectable endotoxins and does not interfere in the test.

Plastics used in the performance of the test are molded, meaning that plastic pellets have been heated to a very high temperature to produce molten plastic in preparation for the molding process. This high temperature will destroy bacterial endotoxins, so molded plastics are free of detectable endotoxins when they are released from the molds. However, depending on the subsequent handling, recontamination is possible. Control measures should be in place to ensure appropriate storage conditions and avoid contact with potentially contaminating substances. However, unless depyrogenated plastics are stored under damp conditions or encounter substances during handling in which GNB could proliferate, the probability of recontamination is remote.

If a laboratory accepts a vendor COA for the endotoxin content of a disposable component, there should be an understanding of the methods used to determine the reported test result. Two examples of COAs are provided below:

• If a shipment of dilution tubes is received from a vendor and is labeled “non-pyrogenic,” what does this really mean? Was the lot tested for pyrogens using a rabbit pyrogen test or a validated monocyte activation test? Was it tested using a standard regulatory requirement and, if so, at what level of endotoxin does the vendor consider the material to be “non-pyrogenic”? It is important to ask the vendor how the material was prepared and tested. 

• If a shipment of 10-mL tubes is received from a vendor with the label of “<0.5 EU/mL,” what does that mean? With what volume was the tube extracted (1, 5, or 10 mL)? A 1-mL extraction would mean <0.5 EU/tube, whereas a 5-mL extraction would mean <2.5 EU/tube and a 10-mL extraction would mean <5 EU/tube. The laboratory should ask the vendor about the method of endotoxin extraction from the tube as well as the BET test conditions that were used to generate the result. 


A default method to confirm the COA result is to treat the plastic disposable as if it were a medical device and proceed according to the methodology provided in USP <161>. However, while this chapter defines the endotoxin limit for medical devices as <20 EU/device, a laboratory may want to consider redefining the limits for disposables used in the test to be less than the value of the most sensitive test used in the laboratory. For example, if the laboratory is using sterile polystyrene tubes for sample dilution, and the most sensitive test is a kinetic chromogenic test with a λ = 0.05 EU/mL, then consider setting a limit low enough to prevent interference across all test methods. 
Likewise, some materials could contain extractable or leachable substances that could inhibit the LAL-bacterial endotoxins assay. The PPC will indicate whether the normal, routine use of the plastic results in any leached inhibitory substance. If a laboratory is considering use of disposable plastic containers such as sterile polystyrene for long-term storage of materials that ultimately will be tested, it is suggested that they test to confirm that there are no inhibitory or enhancing factors that could affect the accuracy of the test. 


When possible, use larger volumes (milliliters) for dilution rather than smaller volumes (microliters), as smaller volumes 
increase variability. 


Standard curves for photometric tests are constructed based on the log10 of the measured onset or reaction times as a 
function of log10 of the endotoxin concentration. Pay attention to the onset times of the standards to ensure that they are consistent from run to run, analyst to analyst, and day to day for any given combination of CSE lot and lysate lot. See Routine Testing, Standard Curve Control, in USP <161>. 


If using a monocyte activation test, be sure to include at least one non-endotoxin control. An analyst requires additional training if any of the following is noted by a supervisor:

• Failure to meet the requirements of the initial performance training.
• Frequent inability to meet system suitability parameters, yielding invalid test results (e.g., confirmation of label claim for 
gel clot, demonstration of linearity for quantitative assays, inability to ensure that negative controls are nonreactive). Note that the inability to recover the PPC within the required range may signal an issue with the analyst’s technique or a change in the product’s manufacturing or formulation that changes the product’s interference profile.

In my next article, I’ll discuss depyrogenation and calculating endotoxin limits and maximum valid dilution.

About The Author:

Yadnyesh Patel earned his master’s degree in microbiology from KBCNM University, Maharashtra, India, in 2009. With over 13 years of extensive experience in quality functions, he has spent time working at pharmaceutical organizations such as Claris Otsuka Ltd, Sun Pharmaceutical Industries Ltd., and Zydus Life Sciences Ltd. Currently, at Zydus group, he spearheads microbiological quality functions, document management, audit, and compliance. He possesses substantial expertise in quality management systems, SOPs, documentation management, microbiological test method validations, sterility assurance, aseptic process simulation, and computer system validation.