Pyrogen Test Expectations for Pharmaceutical Manufacturing

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Mar 24
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Updated: Mar 26

Technical Review Article | Open Access | Published 26th March 2026

Pyrogen Test Expectations for Pharmaceutical Manufacturing

Alessandro Pauletto¹*, Kevin L. Williams¹ | EJPPS | 311 (2026) https://doi.org/10.37521/ejpps31101

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Abstract

Pyrogen control is a critical element of pharmaceutical quality assurance for parenteral products and selected medical devices. Bacterial endotoxins derived from Gram negative bacteria present the predominant pyrogenic risk due to their high potency, resistance to standard sterilization processes, and widespread occurrence in manufacturing environments. Historically, pyrogen testing has relied on in vivo and in vitro methods such as the Rabbit Pyrogen Test (RPT), Bacterial Endotoxins Test (BET), and Monocyte Activation Test (MAT).

The introduction of European Pharmacopoeia chapter 5.1.13 in January 2025 formalized a risk based approach for selecting appropriate pyrogen detection methods. Manufacturers are now expected to perform and document a product and process specific pyrogen risk assessment when justifying exclusive reliance on endotoxin specific testing. This article reviews the principal classes of pyrogens relevant to pharmaceutical manufacturing, evaluates their potency, stability, and likelihood of occurrence, and discusses critical control points and mitigation strategies in sterile injectable processes. A pragmatic framework for implementing pyrogen risk assessment in accordance with Ph. Eur. 5.1.13 is proposed to support robust pyrogen control and patient safety.

Keywords: Pyrogens, endotoxin, pyrogen risk assessment

Introduction

Pyrogens are substances that trigger an endogenous fever response following parenteral administration. They can come from different sources or directly from cytokines released from the immune system¹. They trigger the hypothalamus to raise the body’s temperature “set point.”

The two main types consist of:

Exogenous (from outside the body)

o Bacterial endotoxin (LPS) from Gram-negative bacteria — the most potent/important in pharma QC.

o Other substances from bacteria: lipoteichoic acid (Gram-positive), peptidoglycan, flagellin, toxins (e.g. toxic shock syndrome).

o Viruses

o Non-microbial contaminants: some leachable/extractable from medical devices or packaging can act as pyrogens in humans.

Endogenous (made by the body)

o Cytokines like IL-1, IL-6, TNF-α, and interferons released by immune cells in response to those exogenous triggers.

The activation mechanism can be described very simply, starting from PAMPs (Pathogen-Associated Molecular Patterns)², molecules that are recognized by TLRs (Toll-Like Receptors) or other PRRs (Pattern Recognition Receptors)³. An example of a PAMP is endotoxin, which is recognized by TLR4. The effect of activating a TLR, such as TLR4, is the release of cytokines and prostaglandins (e.g., PGE₂), which ultimately lead to an increase in the basal temperature set by the hypothalamus⁴.

But this is only one of the effects related to endotoxins and other pyrogens. At high concentrations, much more serious events can occur, such as hypotension or edema, platelet aggregation and disseminated coagulation. When associated with massive endotoxin levels, severe conditions such as septic shock can develop⁵.

Fever is therefore only the first warning sign of possible microbiological (and not only) contamination in injectable pharmaceutical products and medical devices.

These products must therefore be verified as apyrogenic or with a pyrogen content below a harmful threshold⁶.

Historically, the tests used in Quality Control (QC) for the detection of pyrogens are:

Rabbit Pyrogen Test: largely being replaced by BET and MAT. Historically, the RPT has been used as a close proxy for the initial occurrence of fever in humans⁷.
Bacterial Endotoxins Test (BET): based on LAL or rFC/rCR reagents specifically for the endotoxin detection
Monocyte Activation Test (MAT): detects endotoxin and non-endotoxin pyrogens (cytokine response)⁹.

It is always important to remember that a product defined as sterile is not necessarily pyrogen-free, since some pyrogens (such as endotoxins) can withstand normal sterilization procedures.

“Sterile” ≠ “apyrogenic.” You can kill bacteria yet still have endotoxin left behind.

Pyrogenicity, demonstration of the absence of non-endotoxin pyrogen (NEP)

In September 2022, EDQM launched a project aimed at the complete elimination of the in vivo rabbit test¹⁰.

The plan foresaw, over a five-year period, the creation of a new chapter in the European Pharmacopoeia (Ph.Eur.) dedicated to pyrogenicity, along with updates to key general monographs (Ph.Eur. 0520, Ph.Eur. 2034, Ph.Eur. 0153, and Ph.Eur. 0125). In parallel, it was necessary to revise chapter Ph.Eur. 5.1.10 (BET guideline) and all product monographs referencing the in vivo rabbit test, namely chapter Ph.Eur. 2.6.8.

In January 2025, chapter Ph.Eur. 5.1.13 on pyrogenicity was published¹¹.

This chapter introduces three fundamental concepts:

1. Endotoxin detection methods: It is possible to use either the LAL test (Ph.Eur. 2.6.14) or the rFC test (Ph.Eur. 2.6.32).

2. Detection of non-endotoxin pyrogens (NEP): The recommended method is the MAT test (Ph.Eur. 2.6.30).

3. Risk assessment: A risk assessment is required to appropriately select the correct detection method to demonstrate the absence of pyrogens in products, choosing between BET and MAT.

This concept of risk assessment is not new, as it reflects what has already been stated in chapter Ph.Eur. 5.1.10 since 2015¹². The main change made to Ph.Eur. 5.1.10 in January 2025 was the transfer of the risk assessment concept to the new chapter Ph.Eur. 5.1.13. Additionally, the following recommendation was added:

"To support the risk assessment, it is recommended to perform experiments with the monocyte activation test together with the validation experiments for the test for bacterial endotoxins using the same batches."

However, this statement is not entirely new, as it has been present since 2019 in chapter Ph.Eur. 2.6.30 regarding the MAT test¹³.

The implementation of chapter Ph.Eur. 5.1.13 has created new challenges for pharmaceutical manufacturers in Europe.

Although the regulation is not retroactive³⁴, it has generated the expectation that a risk assessment must be performed for each new product when the Pharmacopoeia does not explicitly indicate which test to use. In such cases, manufacturers must refer to general monographs, often Ph.Eur. 2034 or Ph.Eur. 0520.

In the past, in the absence of specific guidance on the use of the rabbit pyrogen test, users relied on what was stated in chapter Ph.Eur. 5.1.10, particularly the sentence:

"Although there are a small number of pyrogens that possess a different structure, the conclusion is generally justified that the absence of bacterial endotoxins in a substance or product implies the absence of pyrogenic components, provided the presence of non-endotoxin pyrogenic substances can be ruled out."

However, this part has been removed from chapter Ph.Eur. 5.1.10 as of January 2026.

Therefore, it is now necessary to hypothesise and document a risk assessment each time one intends to use only BET methods for product release, in order to assess the absence of a significant risk of contamination by non-endotoxin pyrogens.

There is a similar situation also for the US for certain specific cases like biological products, where 21 CFR 610.13(b) requires a rabbit pyrogen test¹⁴. This specific requirement may be waived if a method alternative to rabbit pyrogen test meets 21 CFR 610.9 equivalence criteria¹⁵. For human and animal drugs, some USP monographs still require a rabbit pyrogen test, but firms may use an endotoxin or cell-based test if equivalence is proven The appropriate FDA review division will consider alternative methods, such as monocyte activation, on a case-by-case basis.

LPS is the dominant pyrogen in pharmaceutical manufacturing

In the pharmaceutical environment, as well as in medical devices, the focus is mainly on pyrogens of endotoxin nature because they have characteristics of potency, stability, and ubiquity that other classes of pyrogens cannot match.

Let’s compare side by side endotoxins and the main other types of non-endotoxin pyrogens (NEPs), considering these three key factors: potency, stability, and ubiquity.

Potency

Endotoxins (LPS): according to the various BET chapters of Ph. Eur., USP, and JP, the threshold value is defined as 5 EU/kg/hour. This is an activity value and therefore does not represent a unit of weight. The activity of endotoxins varies depending on the Gram-negative species, their growth conditions, and the aggregation state of the endotoxins themselves (and thus also the solution in which they are present)¹⁶,¹⁷. Generally, we can approximate 5 EUs as about 0.5–1 ng of LPS.
Exotoxin: Staphylococcal pyrogenic toxins / superantigens (e.g., TSST-1, SEB) are very potent protein pyrogens (massively amplify cytokines); clinical/experimental contexts show low-ng to pg ranges in plasma detection and strong febrile responses, though “fever threshold dose” in healthy volunteers isn’t ethically defined. Also, Streptococcus exotoxin can stimulate cytokine release from human mononuclear leukocytes¹⁸,²⁰,³⁰
Gram-positive cell wall components (LTA, peptidoglycan): doses to elicit febrile responses are typically orders of magnitude higher than those of exo- and endotoxins (often µg–mg per kg range in animals), and detection often relies on MAT rather than LAL/rBET²¹

➤ Lipoteichoic acids (LTA), as might be found in, for example, Staphylococcus aureus. LTA represents a Gram-positive non-secreted toxin. Pyrogenic dose seems to be at concentrations ranging from 50 µg to over 500 µg²²

➤ Peptidoglycan (PGN), common to both Gram-negative and Gram-positive bacteria (although it is found in much higher quantities with Gram-positive bacteria). Peptidoglycan is a bag-shaped macromolecule that surrounds the cell. Although peptidoglycan is demonstrably pyrogenic, very high numbers of Gram-positive bacteria are required to trigger a pyrogenic response (at around 108 cells); in contrast, with endotoxin, a single Escherichia coli cell contains about 2 million lipopolysaccharide molecules per cell (20). The pyrogenic dose can be different between different animal species but usually ranges between 15-60 ug/Kg²³

Fungal polysaccharides (β-glucans, mannans) can be pyrogenic via dectin/CR3 and other pathways; far less potent than LPS by mass. They can activate LAL (via Factor G) but do not trigger rFC/rCR, and fever generally requires much higher exposures. They could be pyrogenic at a level of 108-109 fungal cells²⁰,²⁴.
Viruses: Several viral species can induce fever responses in humans (e.g., rhinoviruses associated with the common cold). There is no precise universal quantity defined for viral particles to induce fever, because the pyrogenic effect of viruses depends on the type of virus (e.g., influenza, rhinovirus, adenovirus) and viral load (number of infectious particles). No standardised pyrogenic dose for viruses is defined in pharmacopeias or QC guidelines. A potential pyrogenicity could be due to ssRNA by activation of the TLR7 and dsRNA by TLR3²⁵,²⁶. dsRNA is more stable than ssRNA, with which it shares high pyrogenicity values²⁶.

Stability

Endotoxins (LPS): notoriously resistant to sterilization cycles by autoclave, they can potentially be removed by filtration, considering that their molecular weight ranges from about 20 kDa to 1000 kDa depending on the aggregation state. The most widely known removal method is depyrogenation by dry heat, using cycles at 250 °C for at least 30 minutes. Strong chemical treatments (acids/bases) can also have some effectiveness²⁷,²⁸.
Staphylococcal superantigens (SEB/TSST-1): more heat-stable than most cytokines, still far easier to inactivate than LPS (especially by dry heat).
Gram-positive cell wall components (LTA/PGN): non-protein polysaccharide/lipid polymers, heat-stable, chemically removable with strong base/oxidants²⁹
Fungal β-glucans/mannans: very heat-stable polysaccharides, persist through autoclaving; require filtration or aggressive cleaning, but potency is low²⁴.
Virus: When viral contamination is a concern, pharmaceutical manufacturing should incorporate steps for virus removal, such as solvent–detergent treatment or nanofiltration, or inactivation methods such as heat treatment. pH adjustment can also aid in virus elimination. These measures are generally effective in abolishing haemagglutinin activity and, consequently, pyrogenicity²⁰.

Ubiquity

Endotoxins (LPS): virtually everywhere because Gram negative bacteria can be found almost everywhere. Endotoxins could be found in: Purified Water/WFI loops, rinses, stainless surfaces (that hold water that can be evaporated, leaving endotoxin - also applies to rubber stoppers, glassware, tubing/bags, etc.), raw materials handled wet. Endotoxin can stick strongly to surfaces²⁰.
Fungal β-glucans / mannans: potentially could come from cellulose/depth filters, plant-derived excipients (HPMC, cellulose gums, starches), environmental spores/linens/cardboard²⁰.
Gram-positive cell wall components (LTA, peptidoglycan): their presence in pharmaceutical products is generally linked to human sources (e.g., Micrococcus, Staphylococcus aureus), powdered excipients, dry-room/packaging environments, and equipment²⁰.
Staphylococcal superantigens (TSST-1/SEs): these typically originate from human contamination, as in pharmaceutical environments the typical vehicle is the human component¹⁹.

Material and Device-Associated Pyrogens

For implantable and medical devices, pyrogenic risk must also take into account surface-mediated effects. ISO 10993-11 and USP <151> provide guidance for in vivo pyrogen or systemic toxicity evaluation. Extractables and leachable testing, combined with biocompatibility assessment, remains the primary strategy for identifying material-related pyrogens. According to ISO 10993-11, the Monocyte Activation Test (MAT) is currently not validated for material-dependent pyrogenicity, and the use of the Rabbit Pyrogen Test is recommended³¹.

PYROGEN RISK ASSESSMENT

The analysis of pyrogenic contaminants in pharmaceutical preparations allows classification into distinct groups based on origin and structure. This categorization supports risk assessment and the definition of mitigation strategies for each class to ensure control measures are proportionate to the likelihood and impact of contamination. The main methods by which pyrogens can be eliminated can be summarized as follows:

Heating to inactivate heat-labile pyrogens
Extraction processes using organic solvents that dissolve the target compound but not water-soluble pyrogens (if applicable)
Filtration processes to separate substances with higher molecular weight such as proteins or DNA/RNA
Ion exchange chromatography could support in removing residual of ssRNA and dsRNA
pH variations by using strong acids or alkali that may destroy certain pyrogens (if applicable, i.e. target compound is stable)

Additional mitigation strategies can further reduce the risk of pyrogen contamination in finished pharmaceutical products. These include controlling bioburden in raw materials and monitoring endotoxin levels in process water. The diagram below provides a brief (and limited) summary.

A typical manufacturing process for sterile injectable medicinal products is conducted in compliance with Good Manufacturing Practices (GMP) and is based on a series of clearly defined and controlled steps.

The process starts with the preparation of Active Pharmaceutical Ingredients (APIs) and excipients, which are often sourced from qualified external suppliers different from the manufacturer of the finished product. Supplier qualification and incoming material controls are essential GMP requirements. This phase is critical, as appropriate microbiological and quality controls are fundamental to ensuring a manufacturing process with a low risk of microbial and pyrogenic contamination³².

APIs and raw materials are transferred into dedicated preparation vessels, where they are dissolved or suspended under controlled and validated aseptic conditions. The correct execution of Clean in Place (CIP) and/or Sterilize in Place (SIP) for these containers represents a critical control measure for the prevention of microbiological and pyrogen contamination.

Dissolution and suspension activities are performed in classified areas, in accordance with established environmental monitoring programs. At this stage of the process, Water for Injection (WFI) represents a critical element, and its generation, storage, distribution, and point of use must be fully compliant with microbiology specifications.

Following solution or suspension preparation, an initial filtration step is commonly performed using 0.2 µm filters to reduce the bioburden prior to further processing. Filter integrity testing before and after use is mandatory and must be documented in accordance with validated procedures. This step precedes the next critical phase of the process, namely aseptic filling.

The filling operation is conducted under aseptic conditions, typically in Grade A environments achieved through cleanrooms or isolator technology, with Grade B as the background where applicable. Filling is generally performed using validated automated filling lines, designed to minimize human intervention in line with contamination control principles. Subsequent steps include container closure (sealing) and, where applicable, terminal sterilization, which must be validated to ensure sterility assurance without compromising product quality.

The identification of Critical Control Points (CCPs) is essential for the mitigation of microbial and pyrogenic contamination risks. A CCP is defined as a process step at which a specific control can be applied and is essential to prevent, eliminate, or reduce a quality risk to an acceptable level. CCPs must be supported by defined acceptance criteria, monitoring strategies, corrective actions, and proper documentation in accordance with GMP expectations³³

Based on what has been written so far, it is now possible to define a risk assessment specific to the individual product and process for which the BET test is to be implemented in accordance with the requirements set out in Ph.Eur. 5.1.13.

Generally, this risk assessment should consist of the following parts:

1) Introduction: defines the current regulatory framework and the reasons for carrying out this risk assessment. In addition to clearly providing information about the product in question.

2) Summary of pyrogen risks other than endotoxins, possible sources, and feasible mitigation measures.

3) Analysis of the various components involved in the production cycle with regard to their possible contamination by pyrogens and, again, any mitigation strategies. Examples of this include all containers, from primary packaging to containers on the production line, pipes, filters, raw materials (including water, if applicable), and the API.

For products of biological origin, the cell lines used play a fundamental role in risk analysis, as does all material associated with their culture and purification, as well as the corresponding derived products.

4) Conclusions: results of the analyses relating to points 2 and 3

An example of a table relating to point 3 is shown above. Please note that it is included for illustrative purposes only

Conclusion

The ability to implement an effective pyrogen analysis strategy depends entirely on two main factors that we have examined in this short article:

1. A thorough understanding of your products and the associated manufacturing processes, including how these may introduce pyrogens as well as how they can remove them.

2. The ability to understand how different pyrogens can potentially compromise the microbiological quality of pharmaceutical products, contaminating both the manufacturing process and the final product.

With these two points clearly in mind, it will be possible to carry out an appropriate pyrogen risk assessment as defined by Ph. Eur. 5.1.13, thereby ensuring not only the right detection method but also the quality of the finished product and the patient safety

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