Importance of the Endotoxin Standard in the LER Hold-Time Study

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Peer Review Article | Open Access | Published 18th December 2025

Importance of the Endotoxin Standard in the LER Hold-Time Study

Alessandro Pauletto¹*, Christian Faderl², Holger Grallert², Gregory Devulder³, Luca Di Bello², Kevin L. Williams⁴, | EJPPS | 304 (2025) |https://doi.org/10.37521/ejpps30404

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Abstract

Endotoxin detection is essential in biopharmaceutical production to ensure injectable drug safety. PDA Technical Report 82¹ recommends using Control Standard Endotoxins (CSEs) or Reference Standard Endotoxin (RSE) in hold-time studies. However, this study challenges the assumption that all CSEs behave consistently, revealing significant variability in their reactivity compared to RSE.

Simplified matrices (10 mM citrate, PBS, and polysorbate 80) were used to mimic biopharmaceutical conditions while isolating key factors influencing endotoxin reactivity. The study found that the commercially available CSEs tested exhibited inconsistent behaviour, raising concerns about their reliability in endotoxin testing. In contrast, RSE remained stable and reproducible, reinforcing its status as the most well-characterized standard.

This variability may stem from differences in bacterial strain sources, purification processes, and growing conditions, which can lead to endotoxins with distinct structural modifications. These differences may affect endotoxin masking and Low Endotoxin Recovery (LER), leading to inconsistencies in detection.

Given these findings, further standardization of endotoxin testing protocols is necessary. The inconsistencies among CSEs raise questions about their suitability as reliable benchmarks, potentially impacting regulatory compliance and product safety. Re-evaluating the role of CSEs is crucial to ensuring a more robust and reproducible endotoxin detection framework in the biopharmaceutical industry.

Key words: Low endotoxin recovery, LER, CSE, RSE, endotoxin testing, masking endotoxin

Introduction

Endotoxin, or lipopolysaccharide (LPS), is a complex amphipathic molecule that tends to aggregate in aqueous environments². It is an essential component of the outer membrane of most Gram-negative bacteria and is associated with a variety of biological activities, including its strong pyrogenic properties. This potent ability to induce fever and inflammatory responses makes endotoxin a critical contaminant to avoid in parenteral drugs and medical devices.

To mitigate the risk of endotoxin contamination, the Bacterial Endotoxins Test (BET), which employs Limulus amebocyte lysate (LAL) or recombinant Factor C or recombinant Cascade reagents (rFC or rCR), has been integrated into pharmacopeial standards. BET is now the benchmark method for endotoxin detection and is widely adopted in the pharmaceutical and medical device industries. These tests provide a robust and reliable means of ensuring product safety and compliance.

Despite these advancements, specific sample matrices have been shown to mask the ability of these tests to detect endotoxin, particularly in the context of Low Endotoxin Recovery (LER)³. This phenomenon, first described in 2013 by Chen and Vinther⁴, refers to the inability to recover spiked endotoxin from a sample matrix containing chelators and surfactants. Their groundbreaking study revealed that traditional methods, such as dilution or magnesium replacement failed to recover masked endotoxin.

The emergence of Low Endotoxin Recovery (LER) has significant regulatory implications. The US FDA mandates the inclusion of LER data from Hold Time Studies (HTS) in Biologic License Applications (BLAs)⁵. Similarly, since September 2023, the European Medicines Agency (EMA), in its Question-and-Answer document for biological medicinal products, has recommended performing HTS for products containing both a surfactant and a chelator as part of their formulation when submitting a Marketing Authorization Application (MAA). In cases where LER is identified, an appropriate mitigation strategy must be developed and implemented⁶.

However, the definition, identification, and mitigation of LER remain complex and contentious topics in the industry. Central to this debate is the question of which type of endotoxins should be used in these studies⁷. There are two primary categories: Naturally Occurring Endotoxins (NOE) and Standard Endotoxins (RSE and CSE).

Standard Endotoxins, including the Reference Standard Endotoxin (RSE-USP), its European counterpart (Endotoxin BRP), and secondary standards such as Control Standard Endotoxins (CSEs), are the most commonly used in endotoxin testing⁸. These standards are highly reproducible and well-characterized, serving as analytes for validating endotoxin detection methods. In contrast, NOEs are derived directly from bacterial sources and are not subject to standardization. Currently, there is no consensus on the production methods or the bacterial species that would serve as the source for NOEs. This lack of standardization has led to inconsistent results in LER HTS⁹.

Recognizing this, the PDA TR82 recommends the primary use of RSE and CSE for HTS to evaluate the presence of LER. However, even CSEs are not without limitations. They lack uniformity in their defining characteristics, aside from being standardized against a primary reference standard. This variability is compounded by differences between manufacturers, and even between batches from the same supplier.

Such variability raises the possibility that different CSEs could yield disparate results in LER HTS, much like the inconsistencies observed with NOE. This underscores the need for a more standardized approach to both NOEs and CSEs to ensure reliable and interpretable outcomes in LER testing.

As the industry continues to refine its understanding of LER, the development of standardized protocols and reference materials will be critical. These advancements will improve the reliability of endotoxin detection and enhance the safety and efficacy of pharmaceutical and medical device products globally.

Material and methods

Chemicals and materials

Polysorbate 80, PBS (phosphate buffered saline) and trisodium citrate were obtained from Sigma- Aldrich Chemie GmbH, Steinheim, Germany. Endotoxin-free water (ENDOGRADE® WATER) and borosilicate glass tubes (ENDOGRADE® GLASS TUBES) were obtained from bioMérieux SA. The recombinant Factor C test (ENDOZYME II®) was obtained from bioMérieux, Marcy-l’Etoile, France, Limulus amebocyte lysate test (Kinetic-QCL™) was obtained from Lonza Inc., Walkersville, USA

Lipopolysaccharides

For this study, five different Control Standard Endotoxins (CSE) from four different suppliers were used. The study does not specify which CSE was used for each individual test. The CSE included:

Associates of Cape Cod (ACC): Code E0005, derived from E. coli O113:H10.
bioMérieux: Included in the ENDOZYME II® kit, derived from E. coli O113:H10.
Charles River Laboratories (CRL): Codes E110 and E170, both derived from E. coli O55:B5.
Lonza: Code N186, derived from E. coli O55:B5.

Additionally, as a reference standard, the study utilized Reference Standard Endotoxin (RSE) lot HOK354 with 10,000 EU/vial from USP. To determine the endotoxin activities, all different CSEs were measured using a kinetic chromogenic assay (Kinetic-QCL™) and a rFC method (ENDOZYME II®).

Preparation of samples

Six sample stock solutions (matrices) were prepared in 100 mL Schott bottles as follows:

10 mM Citrate + 0.05% Polysorbate 80, pH 6.4
10 mM Citrate + 0.025% Polysorbate 80, pH 6.4
10 mM Citrate + 0.01% Polysorbate 80, pH 6.4
1x PBS + 0.05% Polysorbate 80, pH 7.2
1x PBS + 0.025% Polysorbate 80, pH 7.2
1x PBS + 0.01% Polysorbate 80, pH 7.2

100 mM Citrate or 10x PBS (100 mM phosphate) was mixed with the appropriate amount of 5% Polysorbate 80 and endotoxin free water to reach concentrations as in the stock solutions mentioned above. The individual pH value was adjusted by addition of 6 M HCl and 5 M NaOH solutions. Afterwards endotoxin free water was added to a final volume of 40 mL stock solution. Samples were prepared in 10 mL depyrogenated borosilicate glass tubes, with a sample volume of 5 mL per tube. Unless otherwise specified, samples were spiked with the appropriate concentration of CSE to achieve a final endotoxin concentration of approximately 10 EU/mL. After spiking, samples were shaken at 1400 rpm for 1 minute and for each time point one 0.5 mL aliquot was prepared in ENDOGRADE Glass Tubes. The aliquots were sealed with Parafilm and incubated at either room temperature (20°C incubator) or 4°C for various time periods. A 5 mL water control (endotoxin free water) was also spiked and prepared in the same way as the samples. Endotoxin recovery kinetics were measured using a direct incubation approach.

All samples and water controls were diluted 1:100 in endotoxin-free water before measurement.

Detection of endotoxin

Two different detection methods were used: the kinetic chromogenic assay (Kinetic-QCL™) and the recombinant Factor C assay (ENDOZYME II®). All assays were performed following the supplier’s instructions. For the LAL-based and the rFC-based assay, 100 μL of samples, positive product controls (PPC) and the prepared standard curve dilutions were added in duplicate to a 96-well microtiter plate. A 10 μL endotoxin spike was added to the PPC wells and lysate or reaction mixture was then added.

For the LAL-based method the amount of released chromogenic substrate was measured at 405 nm with a BioTek ELX808 absorbance microplate reader (BioTek Instruments, Inc.) coupled with Gen5 (BioTek Instruments, Inc.) at 37 ± 1 ◦C. The mean of the duplicates per sample was used to determine endotoxin concentrations (EU/mL) using a standard curve fitted with a linear regression model. The detection limit of the assay was 0.005 EU/ mL.

For the rFC assay, the amount of released fluorescence substrate was measured spectrophotometrically at 440 nm with a Synergy HTX fluorescence microplate reader (Agilent Technologies) at 37 ± 1 ◦C coupled with Gen5 software. The mean of the duplicates per sample was used to determine endotoxin concentrations (EU/mL) using a standard curve fitted with a linear regression model. The detection limit of the assay was 0.005 EU/mL.

Results and discussion

Comparison of detection systems

To evaluate the potential impact of two different detection methods on the same matrices spiked with RSE, an initial analytical run was performed. This test compared the six different matrices spiked with only RSE. The chosen hold time points for this study were 1, 2, 3, and 7 days, all at room temperature. The comparison of results, aimed at determining the reduction in detectable endotoxin content, was performed against a water control sample. The control consisted of endotoxin-free water spiked and treated in the same way as the six matrices under examination. Both the samples and the control were diluted 1:100 in endotoxin-free water for measurement. As shown in Figure 1, the overall trend between the two detection methods was highly comparable, showing no relevant differences. For all six matrices, the detected endotoxin levels were below 50% of the initial value (time 0) in the control sample by the seven-day time point. Matrices based on 10 mM citrate exhibited a more pronounced reduction. For these matrices, endotoxin levels were already below the 50% recovery threshold by the first point (1 day). Conversely, for the matrices based on 1x PBS, the endotoxin content dropped below the 50% threshold starting at the two-day time point for those with higher polysorbate concentrations (0.05% and 0.025%). For the 1x PBS matrix with 0.01% polysorbate, the threshold was only crossed at the seven-day time point.

Figure 1: Comparison of different detection methods for CSEs and RSE recovery across six different matrices, evaluated at multiple timepoints during incubation at room temperature.

Temperature effect

As highlighted in several other studies¹⁰,¹¹,¹², incubation temperature can play a significant role in determining different outcomes in hold time studies conducted at different temperatures. To investigate this phenomenon further, the experiment was repeated under the same conditions as before but focused on comparing the effects of temperature (4°C versus room temperature)

In the first case, the comparison was performed across the same time points (1, 2, 3, and 7 days) using the same RSE spike concentration (10 EU/mL). The six different matrices were diluted 1:10 in endotoxin-free water, and the detection method used was the recombinant rFC-based method ENDOZYME II® only. The decision to use only recombinant reagent was based on the results obtained in the previous test “Comparison of detection system” where no relevant difference was found between the two detection systems.

As shown in Figure 2, the results confirm the phenomenon that high variability is associated with various hold time temperatures. At room temperature, all matrices demonstrated a significant reduction in detectable endotoxin content by day 7, falling below 50% of the value observed in the time 0 control sample in water. However, for samples incubated at 4°C, nearly all matrices exhibited values above or very close to the 50% threshold. Notably, matrices based on 1x PBS consistently showed values significantly higher than 100%, relative to the water control sample. In contrast, the 10 mM citrate-based matrices generally displayed values close to the 50% threshold, with slight drops below this threshold at the 2-day time point. No significant differences were observed across the different polysorbate 80 (PS80) concentrations tested.

Figure 2: Temperature effect on LER across six different matrices spiked with RSE at 10 EU/mL. Hold time study conducted over 1, 2, 3, and 7 days at 4°C versus room temperature (RT), using only ENDOZYME II® (EZII) as the detection method. Results are expressed as percentages relative to the time 0 positive control sample in water.

Comparison of RSE against CSEs

To assess whether different Control Standard Endotoxins (CSEs) yield results that differ from those obtained with the Reference Standard Endotoxin (RSE), we conducted hold time studies using the six previously described matrices. These matrices were based on 10 mM citrate and 1x PBS with varying concentrations of polysorbate 80.

The hold time points included were 1, 2, 3, and 7 days. The incubation was performed at room temperature. For detection, the recombinant rFC reagent ENDOZYME II® only was used (based on the assumption of a similar reactivity between the two-detection systems verified in the first experiment)

The results highlight similarities between RSE and CSEs, though with certain exceptions. CSE 1 exhibits reactivity nearly identical to RSE when considering the 7-day time point. Indeed all six matrices yield values below 50% relative to water at T0. However, in the case of CSE 1, this threshold is reached for all matrices as early as the 2-day time point. CSE 3, on the other hand, shows results that are largely comparable to RSE (Figure 3).

In contrast, the results obtained with CSEs 2, 4, and 5 demonstrate notable differences, some of which are significant.

Figure 3: On LER across six different matrices spiked with RSE at 10 EU/mL. Hold time study conducted over 1, 2, 3, and 7 days at 4°C versus RT, using only ENDOZYME II (EZII) as the detection method. Results are expressed as percentages relative to the time 0 positive control sample in water

CSE 2 shows recovery values above the 50% threshold for all matrices up to the 7-day time point, except for citrate-based matrices at higher polysorbate 80 concentrations. The matrix with the lowest polysorbate 80 concentration (0.01%) does not reach this threshold. None of the PBS-based matrices exhibit LER-related issues within 7 days. However, the mean values observed at T0 are higher than those seen with RSE, with recoveries ranging between 150% and 175% for all six matrices. That could be related to an interaction between LPS and the formulation of this CSE with the buffers used in the experiment¹³. Similar behavior is more evident with CSE 5.

Furthermore, while the endotoxin activity in PBS-based matrices remains relatively stable, citrate-based matrices show a pronounced reduction in detectable endotoxin levels over time.

CSE 4 displays a reactivity profile more similar to CSEs 1 and 3 and RSE than to CSEs 2 and 5. All matrices, except for PBS with 0.01% polysorbate 80, exhibit endotoxin levels below the 50% threshold at the 7-day incubation point. Additionally, among PBS-based matrices, only two reach the 50% threshold by the 7th day of incubation, excluding the PBS matrix with the lowest polysorbate 80 concentration.
CSE 5 shows similarities to RSE and CSEs 1, 3, and 4 in citrate-based matrices. In these matrices, endotoxin becomes undetectable as early as the 1-day time point. However, PBS-based matrices show a marked increase in activity at the 1-day time point. This phenomenon may be attributed to an interaction between the specific formulation of CSE 5 and these matrices, leading to a structural change (disaggregation) in the LPS that alters its activity. Notably, this initial structural change is followed by a rapid and significant reduction in activity. The rate of this reduction appears to be partly proportional to the polysorbate 80 concentration.

Conclusion

In this study, simplified matrices were carefully designed and employed to mimic the conditions typically encountered in biopharmaceutical production processes. These matrices comprised 10 mM citrate, PBS, and varying concentrations of polysorbate 80, which are standard and widely used components in many biological drug injectable formulations. While real-world formulations often include additional ingredients or more complex interactions, these supplementary components were deliberately excluded from this study. The objective was to isolate and analyze the primary factors influencing endotoxin reactivity in a controlled manner. Although it is acknowledged that additional components could introduce variability or influence the results to some extent, their absence in this study is unlikely to fundamentally alter the key findings. Instead, this approach provides a focused examination of the core elements and their impact on endotoxin behavior. The results reveal clear and notable variability among certain commercially available Control Standard Endotoxins (CSE) when compared to the Reference Standard Endotoxin (RSE).

As outlined in PDA Technical Report 82, old-time studies have traditionally relied on the use of either RSE or CSE for spiking undiluted samples. These standards are widely regarded as reliable benchmarks for endotoxin testing. The use of Naturally Occurring Endotoxins (NOE) is acknowledged as permissible but is restricted to supplementary studies. This limitation stems from concerns about the inherent variability and inconsistent reactivity of NOE, as highlighted in previous research⁹. Such variability has further solidified the recommendation to prioritize RSE and CSE as the primary standards in hold-time studies.

However, the findings of this study challenge the prevailing assumption that all CSEs exhibit consistent behavior in hold time studies. Significant differences were observed in the reactivity profiles of various commercially available CSEs compared to the RSE. This is particularly concerning given that the RSE is widely recognized as the most well-characterized, stable, reliable, and reproducible endotoxin standard. These discrepancies suggest that the current guidance regarding the use of CSEs in such studies may need to be re-evaluated. The variability among CSEs could undermine the consistency and reliability of results obtained in hold time studies, raising questions about their suitability as reliable benchmarks.

The observed differences in CSE behavior are likely attributable to several factors, including variations in formulation, the bacterial strains from which the endotoxins are derived, the purification processes employed, and the specific growing conditions during production⁹. For example, certain growing conditions may produce endotoxins with distinct structural "decorations" which can alter their susceptibility to the masking effects commonly associated with Low Endotoxin Recovery (LER)¹. These structural differences may render certain CSEs less prone to LER effects, resulting in inconsistencies when compared to the highly standardized and reproducible RSE.

Such findings underscore the importance of further standardization within the field of endotoxin testing. Without uniformity, the variability observed among CSEs could hinder their effectiveness in critical applications, particularly in hold time studies designed to identify and mitigate LER phenomena. A reassessment of the suitability and application of CSEs in comparison to RSE is warranted, as these differences may have significant implications for the reliability and robustness of endotoxin testing protocols. At this stage, it might be more appropriate to rely solely on RSE as a unified standard, enabling global harmonization and standardization of LER studies. This approach would also help to avoid potential inconsistencies in study outcomes caused by the use of different CSEs—similar to the rationale that led to the exclusion of NOEs as a primary standard in PDA TR82

Conflict of Interest

The Authors declare that they are part of a Company providing endotoxin detection reagents and related services

References

1. PDA Technical Report 82, March 2019.

2. Gorman A, Golovanov A. Lipopolysaccharide Structure and the Phenomenon of Low Endotoxin Recovery. European Journal of Pharmaceutics and Biopharmaceutics, Volume 180, November 2022, Pages 289-307.

3. Reich J, Tamura H, Nagaoka I, Motschmann H. Investigation of the kinetics and mechanism of low endotoxin recovery in a matrix for biopharmaceutical drug products. Biologicals, May:53:1-9 2018.

4. Chen J, Vinther A. Low endotoxin recovery in common biologics products. Presented at the PDA Annual Meeting, Orlando, FL. April 15-17, 2013.

5. Hughes P. “Endotoxin – A FDA Perspective.” Presented at the PDA 10th Annual Global Conference on Pharmaceutical Microbiology, Bethesda, MD. October, 19-21, 2015.

6. EMA - Questions and answers for biological medicinal products September 2023 www.ema.europa.eu/en/human-regulatory-overview/research-and-development/scientific-guidelines/biological-guidelines/questions-answers-biological-medicinal-products

7. Bolden J, Knight M, Stockman S, Omokok B. Results of a harmonized endotoxin recovery study protocol evaluation. Biologicals, July:48:74-81 2017

8. FDA Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers (2012)

9. Reich J et al. Low Endotoxin Recovery-Masking of Naturally Occurring Endotoxin. Int J Mol Sci. Feb 15;2019(4):838 2019

10. Reich J, Lang P, Grallert H, Motschmann H. Masking of endotoxin in surfactant samples: Effects on Limulus-based detection systems. Biologicals 2016 Sep;44(5):417-225

11. Tsuchiya M. Factors Affecting Reduction of Reference Endotoxin Standard Activity Caused by Chelating Agent/Detergent Matrices: Kinetic Analysis of Low Endotoxin Recovery. PDA Journal of Pharmaceutical Science and Technology November 2017, 71 (6) 478-487

12. Tsuchiya M. Sample Treatments That Solve Low Endotoxin Recovery Issue. PDA J Pharm Sci and Tech 2019, 73 433-442

13. Parish B. “Design of a Control Standard Endotoxin for Complex Low Endotoxin Recovery Matrices” Presented at USP Modern Methods for Endotoxins and Pyrogens Testing Virtual Workshop. May 15, 2024