The Monocyte Activation Test

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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.).

Definition

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.

How the Monocyte Activation Test method works

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".

Learn about using MAT as a batch release assay

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.

Explore using MAT as a NEP risk assessment

Where does the MAT test apply accordin to Ph. Eur.?

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:

Neisseria meningitidis vaccine

Stoddard, M. B., Pinto, V., Keiser, P. B. et al. (2010)

Hyperimmune

sera

da Silva et al. (2015)
Silva et al. (2018)
Utescher et al. (2018)

Meningococcal
vaccines

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

Studholme et al. (2019)

Tick-borne encephalitis virus vaccine

Etna et al.
(2020)

Yellow fever vaccine

de Mattos et al.
(2018)

Shigella sonnei vaccine

Gerke et al.

(2015)

Rabies

vaccine

Hepatitis B
vaccine

Early adopters of the Monocyte Activation Test

Comparing the Monocyte Activaion Test with other assays

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.

Learn more

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.

Learn more

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

Learn more

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

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

✔ / ✘

✔

✔ / ✘

✘

✔

✔ / ✘

✔

Assay analysis

Common uses of the Monocyte Activation Test among our customers.

Replace the Rabbit Test
As of June 2021, Ph. Eur. is working towards seeing all in-vivo RPT transitioned to in-vitro alternatives (recommending MAT) within 5 years.

Test medical devices by direct incubation
Instead of solely rinsing solutions like BET or RPT, the Monocyte Activation Test detects material-mediated pyrogens by way of direct incubation.

Overcome LER effects
MAT can act as a key tool in helping teams dealing with low endotoxin recovery due to its uniquely low susceptibility to interfering factors.

Use cases of the Monocyte Activation Test

What regulatory bodies have to say about the Monocyte Activation Test

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.).

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.

USP

The United States Pharmacopeia

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

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.

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.

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.

Regulatory bodies on the Monocyte Activation Test

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

✘

✔

✘

✔

Reproducibility of results obtained

Selecting the right Monocyte Activaion Test for you

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/ml, 0.02 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 vials, 450 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.

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.

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.

CTL-MAT kit

Market leading reactivity, sensitivity and reproducibility.

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.

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.

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.