Monocyte Activation Testing parenteral products at batch release.

Parenteral medical devices, biologics, veterinary products and pharmaceutical products require bioburden, sterility and either endotoxin or pyrogen tests (depending on labelling) during their batch release testing process

 

Pyrogen testing a must for many products at batch release.

The unique characteristics of many finished products have regulatory submission bodies mandate them to be tested for both endotoxins and non-endotoxin pyrogens. Examples of such products include medical devices, drugs that themselves impact body temperature regulation, intrinsically pyrogenic vaccines, drugs which stimulate immunological reactions, blood derived products as well as those which interfering factors when tested with LAL assays.

MAT the only recommended in-vitro pyrogen test alternative.

While the Rabbit Pyrogen Test (RPT) has long been the gold standard of the comprehensive pyrogen test during the process of batch release testing, the global transition away from in-vivo assays in pharmaceutical testing at large has led pharmacopoeial bodies like Ph. Eur. to specifically point to the use of the in-vitro Monocyte Activation Test (MAT) as a required pyrogen test, where applicable and after product-specific validation.

What is the Monocyte Activation Test and how does it work?

Very simply, the MAT test is a simulation of the human immune system response to endotoxin, non-endotoxin pyrogenic contamination. Broadly, the test method is carried out as follows:

(i)    Incubation

Your medicinal product sample is incubated with peripheral blood mononuclear cells (PBMC).
 

(ii)   Activation

Overnight, the toll-like receptors (TLR) of the monocytes recognise the presence of pyrogens and activate the signalling pathways that launch the human immune system response. In turn, this stimulates cytokine release.

(iii)  Detection

The next morning, a human IL-6 ELISA is used as a readout to detect and quantify the concentration of released cytokines.
 

(iii)  Analysis

Using the LPS standard curve, the quantified cytokine concentration is finally converted to the Endotoxin Equivalent Units/ml. If below the product's CLC, the MAT is passed.

 

The process of employing MAT as your pyrogen test release assay.

Step 1

Step 2

MAT Product-specific validation
The process of ‘product-specific validation’ is intended to validate the absence of  possible interference between the MAT test and the product being tested. Under Ph. Eur. guidelines, this works by spiking an uncontaminated batch of the product with endotoxin and non-endotoxin pyrogens which is then tested using the MAT. The absence of interfering factors between the product and MAT assay is concluded if across three different dilutions (each below the maximum valid dilution of the specific product), the mean recovery of the spiked endotoxin is within the allowable range of 50–200%. 

 

Routine testing using MAT
The process of routine testing (a.k.a. batch release testing) involves taking a sample from the product batch which is intended to be released and testing it using the three aforementioned dilution factors determined during product-specific validation. Where the detected concentration of pyrogen is below the product’s specific contaminant limit concentration (CLC) and when the spiked LPS recovery percentage is within the allowable range of 50-200%, the product is considered pyrogen free and safe for release.

 

Which MAT method is best suited for my product at batch release testing?

The European Pharmacopeia (2.6.30.) outlines three different methods for the Monocyte Activation Test (MAT) during the batch release of products. 

METHOD A:
Quantitative test

Involves comparing the IL-6 release of a non-spiked product preparation with the LPS standard endotoxin dose response curve to determine the concentration of endotoxin equivalent units of contamination present in the product preparation. When lower than the product’s CLC, the MAT is passed.

METHOD B:
Sem-quantitative test

Employed if non-linearity between dose response curve and standard endotoxin curve. Involves comparing IL-6 release of a spiked product preparation with equivalent points of LPS standard preparation. Here, the concentration of endotoxin equivalent units of contamination present in the product preparation is equal to the difference between that of the spiked and non-spiked product preparations. If lower than its CLC, the MAT is passed.

METHOD C:
Ref. lot comparison test

For products that are intrinsically pyrogenic. The contaminant concentration (in endotoxin equivalent units) present in the product preparation is compared with that of a reference lot. If  lower or comparable to reference lot, the MAT is passed.

Benefits of using the Monocyte Activation Test as your batch release assay

Get a head start on Ph. Eur. mandated RPT transition

In June 2021, the Ph. Eur. Commission announced plans to completely replace the rabbit pyrogen test (RPT) in the Ph. Eur., within approximately 5 years. There are 59 Ph. Eur. texts for vaccines for human use, blood products, antibiotics, radiopharmaceuticals and containers which specify the use of RPT and will thus be expected to transition to an in-vitro alternative in the next five years. The Ph. Eur. specifically recommends the use of MAT as a replacement.

Suitability to all product categories

The rabbit pyrogen test is suitable for testing general pharmaceuticals and biologics, while it fails as an assay for cell therapeutics and cannot be applied to all medical devices. The LAL and rFC are also effective for general pharmaceuticals but their enzymatic nature means they cannot be counted on for all medical devices, biologics or cell therapeutics. It is only the Monocyte Activation Test that is suitable and successful when batch release testing any product category

Reflects human biological reaction

The unique characteristics of many finished products have regulatory submission bodies mandate them to be tested for both endotoxins and non-endotoxin pyrogens. Examples of such products include medical devices, drugs that themselves impact body temperature regulation, intrinsically pyrogenic vaccines, drugs which stimulate immunological reactions, blood derived products as well as those which interfering factors when tested with LAL assays.

 

"But what will we need at our site to get started with our MAT testing?"

Check out our neat overview of all the supporting materials, equipment and reagents you'll need on your end to make sure you're MAT battle-ready.

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Want to get free advice from the experts who've already onboarded the world's biggest pharma to MAT?

Simply reach out to us and we’ll schedule an online consultation with our team of scientists. Together we’ll devise a plan to successfully test your medical device – whatever it may be – for material-mediated pyrogens