Sustainable Pyrogen Testing with PyroCell^® Monocyte Activation Tests

Monocyte Activation Test - Assay Principle

With Monocyte Activation Tests, the response of the human innate immune system to pyrogens is measured. The assay uses human monocytes, the key cells of innate immunity, that respond to the presence of endotoxins or non-endotoxin pyrogens by secreting pro-inflammatory cytokines such as interleukin-6 (IL-6) linked to the fever reaction in humans. With PyroCell^® Monocyte Activation Tests, the released cytokines are quantitated using a human IL-6 ELISA assay.

PyroCell^® Monocyte Activation Test Systems are optimized and validated reagent kits for the sensitive detection of pyrogens in pharmaceutical preparations. The PyroCell^® MAT System, IL-6 consists of a cryopreserved peripheral blood mononuclear cell (PBMC) preparation pooled from four human blood donations (pMAT cells), and an optimized, FBS-based Culture Medium Supplement. It has been validated with the Pelikine compact human IL-6 ELISA kit, ensuring robust and reliable results.

The PyroCell^® Monocyte Activation Test, Human Serum System (PyroCell^® MAT HS System) uses the same PBMC preparation and the human IL-6 ELISA detection system but Human Serum as culture medium supplement.  For Monocyte Activation Tests, the use of human serum provides a more sustainable reagent formulation and moreover, has been demonstrated as advantageous for pyrogen testing of blood-based products. On day one of the experiment, the cryopreserved pMAT cells are thawed and incubated with the test substance in a cell culture step (18-24 hours). On the following day, the cell culture supernatants are harvested and the IL-6 cytokine released by human monocytes is detected with the human IL-6 ELISA assay, and measured in an absorbance reader. Using reference endotoxin dilutions, the OD values are finally converted into Endotoxin Equivalent Units (EEU/mL). 

Monocyte Activation Test - Applications

Early detection of pyrogens in a pharmaceutical preparation is critical for quality control programs and an essential product safety measure. Pyrogens may originate from microorganisms such as bacteria and fungi and also from viruses, genome components or other organic particles from the environment. Pyrogens initiate inflammatory reactions when injected into a patient. With the evolution of manufacturing technologies that deliver for example, vaccines, bioprocessed proteins or cell and gene therapy products, new contamination risks may arise in production. Though the Monocyte Activation Test principle was established decades ago, its adoption for life-saving biologics has only accelerated recently. 

PyroCell^® Monocyte Activation Test Systems reflect decades of experience in developing and manufacturing specialized MAT components thus providing the perfect solution for life-saving drugs that are unethical to test in experimental rabbits or cannot be tested with standard bacterial endotoxin tests. 

Qualified cells

• Ethical cell sourcing - blood donations specifically for the Monocyte Activation Test
• Validated process – ensure endotoxin-free preparations in a validated time frame 
• Naïve cells - demonstrated with quality certificates for critical parameters 

Cryopreserved donor pools

• Available on demand - no cell isolations prior to each experiments
• Large lot size prepared with equal cell quantities of 4 blood donations 
• Proven robustness and reliability

IL-6 as cytokine read-out

• Fully secreted into the medium
• Stable in (frozen) supernatant
• Ensure highly sensitive detection

Regulatory Information 

Compliance to regulations requires QC testing of all injectable pharmaceuticals and medical devices for the presence of pyrogens whose effects may range from fever, shock, and in some instances, death. Globally harmonized tests to detect pyrogenic substances are the animal based rabbit pyrogen test (RPT), as well as the bacterial endotoxin test (BET).

The world’s increasing concerns with ethics of using experimental animals, and efforts to protect natural resources led regulatory agencies and pharmaceutical companies to acknowledge in vitro test systems such as the monocyte activation test (MAT) and the recombinant factor C test (rFC) that are minimizing such dependencies. Likewise, the European Pharmacopeia has adopted the MAT in 2009 (Ph. Eur. chapter 2.6.30) and recently, has put an end on the RPT for the year 2026. Other leading pharmacopeia such as the United States Pharmacopeia are describing the MAT as a suitable alternative test method to the RPT.

Other advantages of the MAT include the stimulation of a human response (RPT: mammal response), introduction of experimental controls and cost savings. Therefore, the MAT is closing the gap between patient safety and other regulatory requirements.

Sensitive, robust, reliable - Benefits of PyroCell^® Monocyte Activation Test System

• Patient safety – detection of the full range of pyrogens in one test
• No experimental rabbits – acknowledged by regulators to replace the rabbit pyrogen test
• Human-specific – mimics the innate human response to pyrogens
• Flexible – 2 validated system kits meeting product-specific and sustainability demands
• Standardized – robust application and assay controls meet high sensitivity for reliable results