Endotoxin and pyrogen test service

LER (low endotoxin recovery) was first reported in 2013 (Chen et al. 2013) and describes the observation that endotoxin is underestimated due to masking in various drug product formulations with time. Reports exist where spiked endotoxin in drug formulations was not detectable by LAL but were pyrogenic in rabbit pyrogen test.

Typical formulations comprise a protein component, polysorbate, citrate or phosphate. The effect is strongly dependend on storage temperature, duration of storage, nature of the API and source of spiked endotoxin.

A LER hold-time study performed in our lab includes a detailed project plan and report. The experimental setup will be aligned with your specific needs. The turnaround time depends on the duration of the experimental phase but we guaranty short processing time for preparation of the study and generation of  the report (in general 4 weeks).

As reported by the FDA, classical endotoxin test methods (LAL) can fail to detect masked endotoxin in various drug formulations (low endotoxin recovery).

In 2013 Hyglos GmbH launched a sample preparation kit for the demasking of endotoxin in biopharmaceuticals, EndoRS(1). The new method is based on the combinatorial use of the kit components for the screening for the best demasking mixture. The kit has to be applied in combination with EndoLISA detection method.

Pyrogens produced by gram negative bacteria, i.e., the endotoxin (LPS), are of significance to the pharmaceutical industry (1). Endotoxin ingress is not only due to contaminations. Endotoxin can also occur as a result of the biotechnological process (e.g., E.coli expression system). Especially for such processes, dedicated endotoxin removal steps need to be clearly identified and validated. Thereby, endotoxin removal procedures can be challenging as high and also low levels of endotoxin need to be removed and the product shall remain without alteration.

(1) https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/inspection-technical-guides/bacterial-endotoxinspyrogens

Endotoxin and pyrogen testing is mandatory during process development, as in-process control and for product release in pharmaceutical and medical device industry. In research (cell culture experiments and animal trails) contaminating endotoxins and other pyrogenic substances induce severe physiological reactions which often lead to false interpretation.

The Monocyte acitivation test (MAT) is a cell-based assay: Toll-like receptor mediated stimulation of blood monocytes to produce pro-inflammatory cytokines in response to various pyrogens. The level of released cytokine is determined by a classical ELISA.

We offer quality assurance to laboratories working with Monocyte Activation Test (MAT). Based on our expertise we provide frequently audits of MAT test proficiency. Our Proficiency Test Program (PTP) confidentially verifies working procedures, method and report validation, identifies trends and potential demands for training. This supplies additional value to internal quality assurance.

This assay uses a specially treated limulus amebocyte lysate (LAL) where the Factor C stimulation pathway is inhibited. Therefore only activation by ß-glucan via Factor G leads to activation of proclotting enzyme and conversion of the chromogenic substrate.

Novel therapeutic antibodies can cause severe responses and ultimately lead to life-threatening systemic release of cytokines – termed cytokine release syndrome (CRS) – as seen in a Phase I clinical trial following the administration of the CD28 superagonist antibody TGN1412.1 This demonstrates that (although in rare cases), infusion related responses in patients may only become apparent in first-in-man trials.

In 2020, the FDA released its Nonclinical Safety Evaluation of the Immunotoxic Potential of Drugs and Biologics emphasizing the use of “human cells before initiating clinical trials” as an “an assessment of the potential for cytokine release syndrome caused by therapeutic proteins using unstimulated human cells in both plate-bound (or other assays that can assess the contribution of crosslinking of receptors) and soluble formats with appropriate positive and negative controls”.

Microcoat’s Cytokine Release Assay

• Standardized
• Controls included
• Cohort of 30 healthy donors

Microcoat provides consulting service in the field of pyrogen and bacterial endotoxin testing. Our team is specialized in the implementation of alternative test methods and provides advice in the case of test interference. For the analysis of the Low Endotoxin Recovery (LER) phenomenon, we provide dedicated guidance in planning, performing and interpreting of hold-time studies. Always giving you advice and quick support if you are facing unexpected trouble during execution of your studies. Furthermore, we support you in the development of strategies for mitigation of LER-affected products according to regulatory requirements.