What is the Monocyte Activation Test?

Employing human blood to simulate the first stages of the human immune system, the Monocyte Activation Test (MAT) is an in-vitro assay designed to test parenteral drugs, biologics and medical devices for all classifications of pyrogens. While for 100 years the Rabbit Pyrogen Test has been the gold standard for pyrogen testing, regulatory bodies like the European Pharmacopeia (Ph. Eur.) are now mandating the transition towards in-vitro alternatives – specifically recommending the Monocyte Activation Test where possible and after product specific validation (2.6.8.).

 

How does the Monocyte Activation Test method work?

Step 1

Incubate parenteral product in PBMC
A sample of your parenteral product or medical device batch is incubated with Peripheral Blood Mononuclear Cells (PBMC) included in the Monocyte Activation Test kit.

Step 2

Immune system activation
The toll-like receptors of the monocytes are given time overnight to detect any pyrogens that may be present in the sample preparation, and if there are any, will launch the human immune system response which activates cytokine release.

Step 3

Quantification of cytokine concentration
A human IL-6 ELISA (included in the Monocyte Activation Test kit) is employed the next morning as a readout to quantify the concentration of released cytokines in the product preparation over the previous night.

Step 4

Endotoxin Equivalent calculation
Making use of the LPS standard curve, the measured cytokine concentration is then converted to the Endotoxin Equivalent Units/ml understood to be present in the product. 

 
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Where does the Monocyte Activation Test apply according to the Ph. Eur.?

The batch release pyrogen test for parenteral products

The Ph. Eur. General Chapter "Pyrogen Test" (2.6.8) stipulates that "wherever possible and after product-specific validation, the [Rabbit] Pyrogen Test is replaced by the Monocyte Activation Test" as a batch release pyrogenicity assay for parenteral products which are labelled "pyrogen free".

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A non-endotoxin pyrogen "risk assessment" where BET is used

For products employing BET as a release assay, the Ph. Eur. (5.1.10) stipulates that the risk of non-endotoxin pyrogenic substances must assessed and ruled out while the product's production process is being established, or upon batch release once it has been established by way of a Monocyte Activation Test.

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Early adopters of  the Monocyte Activation Test

Over the last 5 years, vaccines which previously used the Rabbit Pyrogen Test as their release assay have been among the very first to take on the Monocyte Activation Test (MAT). Unlike MAT, Bacterial Endotoxin Tests are often unsuitable for products that are intrinsically pyrogenic or those that include additives commonly included in vaccines like aluminium hydroxide which tend to interfere with the assay (Hartung, 2021). Here are some examples of MAT's uptake in testing vaccines:

Meningococcal
vaccines

Vipond et al. (2016, 2019) Valentini et al. (2018)

 Studholme et al. (2019)

Tick-borne encephalitis virus vaccine

Yellow fever vaccine

Shigella sonnei vaccine

Rabies

vaccine

Hepatitis B
vaccine

 
 

The Monocyte Activation Test vs. other pyrogen and endotoxin tests

Human-specific, In-vitro 

& most sensitive

The Monocyte Activation Test is the only endotoxin or pyrogen assay that is in-vitro, reflects the human immune system and with an LoD of 0.004 EU/ml, it's the most sensitive.

Detects broadest range of pyrogens

Only the Monocyte Activation Test  can detect all pyrogen classifications including those deriving from gram positive and gram negative bacteria, as well as Yeasts, Molds and Viruses.

Optimised for the most product categories

Where BET or RPT sometimes fail, the Monocyte Activation Test is successful in testing all products from medical devices, therapeutics, pharmaceuticals & biologics

Assay analysis

Limulus

Amebocyte Lysate Test (LAL)

Rabbit

Pyrogen Test (RPT)

Recombinant factor C
(rFC)

Monocyte Activation Test (MAT)

Assay type

Sensitivity (Limit of Detection)

Delivered

results

Ph. Eur. perspective

Ex-vivo

In-vivo

0.05 EU/ml

0.005 EU/ml

Quantitative Qualitative

Qualitative

Quantitative

Quantitative

Inadequate for pyrogen testing

Being

transitioned

out

In-vitro

0.005 EU/ml

Inadequate for pyrogen testing

In-vitro

0.004

Approved

Detectable pyrogenic contaminations

Endotoxins (from
Gram negative
bacteria)

Yeasts & Molds

Gram-positive
bacteria

Viruses

/ 

Possible applications

Pharmaceuticals

Biologicals

/ 

Medical devices

Cell therapeutics

/ 

/ 

/ 

/ 

/ 

 
 

What regulatory bodies have to say about the Monocyte Activation Test

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PH. EUR. / EDQM

The European Pharmacopoeia

Recommends the Monocyte Activation Test as RPT replacement wherever possible after PSV (2.6.8.); recommends MAT as a NEP risk assessment (5.1.10.). 

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FDA

The United States Food and Drug Administration

In 2009 notes, detailed that the Monocyte Activation Test may be used for parenteral products once PSV carried out.

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USP

The United States Pharmacopeia

"Pyrogens" General Chapter <151> permits using a "validated equivalent in-vitro pyrogen or bacterial endotoxin test" where appropriate.

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ISO

The International Org. for Standardization

Whenever found to deliver equivalent info. of relevance, ISO 10993-1:2018 expresses preference for in-vitro rather than in-vivo models.

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IPC

The Indian
Pharmacopoeia

The Monocyte Activation Test was included on 1st July 2018 in IPC's 8th edition as an in-vitro alternative to the Rabbit Pyrogen Test.

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JP

The Japanese Pharmacopoeia

According to JP XVII, validation of alternative methods like the Monocyte Activation Test is possible if delivers better precision and accuracy.

 

What Monocyte Activation Test kits are available out there?

There are primarily two groups of Monocyte Activation Test currently available on the market. These are distinguished by the type of cell source they employ as the canvas of monocyte activation. The first group makes use of the cell-line mono-mac-6 as its cell source, while the second employs cryopreserved donor-pooled peripheral blood mononuclear cells (PBMC). The table below offers a high level overview of the key strengths and weaknesses of each.

Cell-line
mono-mac-6

(MM6)

Peripheral blood mononuclear cell (PBMC)

Ph. Eur. description on NEP detection

Risk of donor variation

Reflects behaviour of healthy donor

Stable expression of TLRs

"limited"

"suitable"

Low

Low

Reproducibility of results obtained

 

Factors to consider when  choosing the right Monocyte Activation Test for you.

01

MAT cell source
There are currently two commercialised Monocyte Activation Test cell sources available globally: (i) Mono-Mac-6 (MM6) cell-line and (ii) Peripheral Blood Mononuclear Cells (PBMC). 

The MM6 derives from the blood of a single acute monocytic leukemia patient and as a result the the monocytes have been known to sometimes not have TLRs that reflect the stable expression required to consistently detect pyrogenic contaminants and initiate the release of cytokines as a healthy human's would. Resultantly, the reproducibility of MAT results have been found to be low using this cell source. The Ph. Eur. (2.6.30) also describes MM6 based MAT kits as "limited" in their ability to detect non-endotoxin pyrogens.
 

PBMC based MAT kits source their PBMC from the pooled blood of screened, healthy donors which means when incubated with a spiked product sample, the process of monocyte activation can be counted on to reliably simulate that of a healthy human. As a result, results for MAT kits based on this cell source have been consistently found to be reproducible. The Ph. Eur. (2.6.30) counts this cell source as proficient in detecting both endotoxins and non-endotoxin pyrogens.

02

Assay sensitivity
The "sensitivity" of a Monocyte Activation Test kit describes its limit of detection (LoD). The lower the limit of detection, the more sensitive the MAT kit is. While until recently the sensitivity of an assay wasn't too much of an important factor to consider, picking a Monocyte Activation Test with a very low limit of detection can be make or break when you're going through product-specific validation for a product that has either a low contaminant limit concentration (CLC) or there are interfering factors between the product and the MAT assay. Learn more here.

Besides
MAT BioTech's CTL-MAT kit, There are currently 3 other commercialised PBMC-based Monocyte Activation Test vendors on the market. Each has an LoD of 0.125 EU/ml0.02 EU/ml and 0.016 EU/ml. The CTL-MAT assay has a market leading LoD of 0.004 EU/ml, making it the most sensitive Monocyte Activation Test available, anywhere.

03

MAT PBMC supply scale
Until now, one of the primary issues faced by pharmaceutical manufacturers when employing the Monocyte Activation Test has been the size of the MAT PBMCs available. The larger the supply of PBMC batches, the less there will be the need to go through revalidations of the PBMC when a new PBMC batch is being used and the more consistency comparability between MAT results across different tests, manufacturing sites and time more broadly.


As already mentioned, besides our CTL-MAT kit, There are currently 3 other commercialised PBMC-based Monocyte Activation Test vendors on the market. Each has a PBMC batch supply size of of 200 vials450 vials and 250 vials. The CTL-MAT assay has a market leading PBMC supply scale of 2,000 PBMC vials per batch, making it by far the most scalable Monocyte Activation Test worldwide.

Monocyte Activation Test cell source cri

The CTL-MAT Monocyte Activation Test kit promises what others simply can't.

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0.004

EU/ml LoD

The lowest limit of detection

The CTL-MAT Monocyte Activation Test kit detects contaminations in products with low endotoxin specifications that other kits can't.

2,000

Kits per batch

Largest Monocyte Activation Test batches

Lets you always make sure your test samples undergo stable and consistent conditions across different trials and validation stages. 

15

Years experience

Longest Monocyte Activation Test history

Call upon a team comprising the very first pioneers of MAT. Whatever your challenge, we've already seen and overcome it.

 

Market leading reactivity, sensitivity and reproducibility.

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Highest reactivity & sensitivity

  • 0.5 EU/ml LPS can produce up to 10,000
    Picogram/ml IL-6.

  • The LLoD of the international endotoxin standard LPS E. coli is determined as (or equal to) 0.004 EU/ml​.

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Highest intra-batch reproducibility

  • The above visualises the standard curve across 4 vials from the same batch.

  • As illustrated, our MAT kit promises almost perfect reproducibility within each batch​.

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Highest inter-batch reproducibility

  • The diagram above visualises the standard curve across vials from 4 different batches.

  • As illustrated, our MAT kit promises almost perfect reproducibility across different batches​.

 
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Unlike any other manufacturer, we provide market leading results across batches of up to 2,000 monocyte activation test kits at a time. This owes to our robotised large scale production facilities. Such a scale is crucial in being able to ensure test samples undergo stable and consistent conditions across different trials and validation stages. 

kits per batch of 4-donor
pooled PBMC

2,000

 

Our Monocyte Activation Test kit includes everything you

need to get started.

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Pooled PBMC

1 ml (5 million cells)

of cryopreserved

4 or 8 donor pooled PBMC.

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EDQM Endotoxin Standard

International endotoxin standard Lipopolysaccharides E. Coli (LPS).

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2 x Non-Endotoxins

Includes both heat-killed Staphylococcus aureus (HKSA) and R848 (Resiquimod).

Media A & B

Culture media to

dilute standards and products.

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Human IL-6 ELISA Kit

Contains all reagents, coated microplate and IL-6 standard.

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96-Well Microplate

Culture plate for incubating product, standard and PBMC.

 

Our five star support team will be there, every step of the way.

Whether or not you're using our Monocyte Activation Test kit, our support team would be happy to help you set-up your Monocyte Activation Test laboratory to deliver robust and reliable results like never before.

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Get in touch with us

for a quote.

Simply fill out our contact form and one of our team will reach out to you

to understand what your need is and give you a price quote.