Assessing Non-endotoxin Microbial Pyrogens in Relation in

Pharmaceutical Processing
By Tim Sandle, Ph.D. Mar 31, 2015

INTRODUCTION
Microorganisms such as Gram-negative or Gram-positive bacteria,
viruses, and fungi contain components that are pyrogenic and which activate the innate immune
system. Pyrogens (a variant of the Greek word pyros, meaning fire), can occur independently
of viable microorganisms. With pharmaceuticals, pyrogens are a major safety concern in
parenterally administered drugs, since they can cause severe reactions such as fever, rash,
headache, myalgia, nausea, vomiting, organ failure, and endotoxic shock to the recipient (1).
In addition to pyrogens of microbial origin, some chemicals act as non-microbial pyrogens.
These are not addressed to any detail in this article. Of the pyrogens of microbial origin, the
pyrogen that has been discussed in the greatest detail is bacterial endotoxin (derived from the
cell wall of Gram-negative bacteria in the form of lipopolysaccharide).
Endotoxin is examined using the Limulus amebocyte lysate test. This is an
established and relatively mature test and one which can be applied to the examination of water
and to most types of finished products.
Aside from endotoxin, one important issue that pharmaceutical
manufacturers of parenteral drugs need to consider is the risk posed by pyrogens of microbial
origin that are not related to lipopolysaccharide. This article assesses microbial pyrogens and
discusses those that may be of a concern to certain types of pharmaceuticals. This is an area
sometimes called into question by regulators and assessors and this can only be answered
through process review and risk assessment.
Currently, there are three testing possibilities available to test for pyrogens: the
Rabbit Pyrogen Test, the Limulus Amebocyte Lysate (LAL) test (Bacterial Endotoxin Test),
and test systems using human whole blood or human monocytes, called Monocyte Activation
Test (MAT). Of these, the LAL test is generally specific only for bacterial endotoxin (although
lysates, unmodified, will react with beta glucan). The rabbit test is a near hundred-year-old
test, mimicking the human response to pyrogens above a certain threshold. The MAT is based
on the human fever reaction and thus most closely reflects the human situation. If there is
pyrogen contamination, the endogenous pyrogen interleukin-1 is released, which is then
determined by ELISA (Enzyme-Linked-Immunosorbent Assay). Whether the rabbit test or the
MAT are needed as quality control tests depends upon the likelihood of other pyrogens in
sufficient numbers.

PYROGENS
Pyrogens can be divided into two classes: exogenous pyrogens,
such as endotoxin from Gram-negative bacteria that induce fever when applied intravenously;
and endogenous pyrogens that are induced inside the body as a reaction to the contact with
exogenous pyrogens and which cause an elevation in body temperature (endogenous pyrogens have potent
pyrogenic and inflammatory activities and include interleukin 1-a (IL-1a), interleukin-1b (IL-
1b), tumor necrosis factor a (TNF-a) and interleukin-6 (IL-6)) (2). For this article, exogenous
pyrogens are the concern. Pyrogens of microbial origin are metabolic products of
microorganisms. Chemically these are lipid substances associated with a carrier molecule,
which is usually a polysaccharide. The carrier may also be a peptide. Pyrogens are produced
by many microorganisms including bacteria, yeasts, and molds. The most potent pyrogens are
the endotoxins produced from the cell walls of the Gram-negative bacteria
(lipopolysaccharide). Generally, pyrogens have a high molecular weight, often, more than
1,000,000 Daltons (3).
Pyrogens trigger physiological effects in slightly different ways. When cells
of the immune system, primarily blood monocytes and macrophages, come into contact with
pyrogens they release mediators transmitting the fever reaction through the organism to the
thermoregulatory centers of the brain. To illustrate this, consider endotoxin:
Endotoxin pyrogen enters the bloodstream.
It binds to lipopolysaccharide (LPS) binding protein (LPB).
LPB takes it to the reticuloendothelial system (RES).
The receptor cells in the RES are the circulating mononuclear cells.
This attachment to the receptor cells causes the production of pro-inflammatory cytokines.
These cytokines are interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-a
(TNFa).
These factors produce inflammation and fever (4).
Similarly, purified Escherichia coli endotoxin (used as a reference standard for the LAL test),
Gram negative bacteria and Gram positive bacteria induce IL-6 secretion by whole blood
cultures (5)
In terms of the nature of pyrogenicity, pyrogenic reactions and shock are induced
in mammals upon intravenous injection of endotoxin at low concentrations (from 1 ng/mL) (6).
The maximum level of endotoxin for intravenous applications of pharmaceutical and biologic
products is set at 5 endotoxin units (EU) per kg of body weight per hour by each of the major
pharmacopoeias (7). The term EU describes the biological activity of an endotoxin. For
example, 100 pg of the standard endotoxin EC-5 and 120 pg of endotoxin from Escherichia
coli O111:B4 have activity of 1 EU (8).

Endotoxin
The most frequently encountered pyrogen in pharmaceutical processing is
endotoxin. Structurally lipopolysaccharides are part of the cell wall of Gram-negative bacteria.
They are composed of a hydrophilic polysaccharide moiety, which is covalently linked to a
hydrophobic lipid moiety. LPS from most species is composed of three distinct regions: the O-
antigen region, a core oligosaccharide and Lipid A (LipA). The lipid A is the most conserved
part of endotoxin and is responsible for most of the biological activities of endotoxin (that is
its pyrogenicity.) (9) The reason for the high prevalence of endotoxin in pharmaceutical
facilities is a result of the use of large volumes of water in processing (water is a key ingredient
to many products and it is also used for equipment rinsing.) The microorganisms found in
association with water are Gram-negative bacteria (such as Pseduomonads). Although
endotoxins are linked within the bacterial cell wall, they are continuously liberated into the
environment. The release does not happen only with cell death but also during growth and
division. Since bacteria can grow in nutrient poor media, such as water, saline, and buffers,
endotoxins will be found at high levels in such environments (with the exception being highly
purified forms of water like Water for Injection).

Other Bacterial Pyrogens

Aside from endotoxin, there are other forms of non-endotoxin microbial
pyrogens. The degree to which these are prevalent in pharmaceutical environments (or rather
prevalent at levels sufficient to trigger a pyrogenic response) is a point open to discussion. In
examining other microbial pyrogens, beginning with Gram-positive microorganisms, here the
pyrogenic component is lipoteichoic acids (LTA), as might be found in, for example,
Staphylococcus aureus. LTA represents a Gram-positive endotoxin that is a non-secreted toxin
(which means, like endotoxin, it is not secreted through normal cellular function and is instead
released through cell lysis. Exotoxin, outlined below, is a secreted toxin.) There is some debate
as to the extent that LTA itself is immunostimulatory, and thereby pyrogenic, or whether it
requires the aid of other proteins (10). Another cell-related material that can be pyrogenic is
peptidoglycan, 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 (11, 12); in contrast, with endotoxin, a single Escherichia coli cell contains
about 2 million lipopolysaccharide molecules per cell.
There is a longstanding debate as to whether peptidoglycan itself can
trigger a response using the LAL assay. If the molecule can elicit a response then considerably
more of the molecule is required to do so when compared with lipopolysaccharide. With this,
one study showed 400µg per mL of peptidoglycan sourced from bacteria of Streptococcus
group A compared with only 0.0005 µg of purified endotoxin was required to produce an
equivalent LAL potency of 0.03 Endotoxin Units (13)). Thus the activity of peptidoglycan is
somewhere between 1,000 to 400,000 times less than that of Escherichia coli
lipopolysaccharide (14). These observations again infer that high numbers of bacteria would
need to be present and lysed. Such observations suggest that good bioburden control can guard
against such a non-endotoxin pyrogen risk. Other pyrogenic substances are bacterial exotoxins,
for example secreted from Streptococcus (15). Exotoxins can trigger toxic shock-like syndrome
(TSLS), characterized by hypotension or shock, fever, multiorgan system involvement. Among
the best characterized exotoxin are superantigens. These are the family of pyrogenic exotoxins
produced by Staphylococcus aureus and Streptococcus pyogenes (16). The related routes of
infection are primarily food poisoning (via ingestion). Although exotoxins have a degree of
heat resistance, this is lower than endotoxin. Therefore any risks to contaminated glassware
intended to be sterile from Gram-positive microorganisms are eliminated through
depyrogenation cycles for glass vials. Another bacterial source comes from enterotoxins, such
as from Staphylococcus aureus. Enterotoxins are single-chain globular proteins of varying
molecular weights. It is not clear the level of enterotoxins needed to elicit a pyrogenic response,
although the levels are again estimated to be considerably higher than endotoxin (at 1 µg/Kg)
(17). Monitoring for high levels of staphylococcal bioburden (especially Staphylococcus
aureus) would act as a riskcontrol in pharmaceutical manufacturing. Thus with peptidoglycan
molecules, exotoxins, and enterotoxins, research suggests that concentrations several orders of
magnitude higher are required to produce similar pyrogenic responses to endotoxin (18).

Fungi and Viruses

Fungi can produce pyrogenic reactions. While this may depend on the type
of fungi, numbers of fungal cells approaching 108or 109 are required to trigger a response (19).
As with bacteria, bioburden and environmental monitoring assessments can guard against the
risk of significant levels of fungi affecting products. Another source of pyrogenic response
comes from viruses. Many types of virus can elicit a pyrogenic response in people (for example,
rhinovirus infections as demonstrated with the common cold). Where viruses are a risk,
pharmaceutical processes need to have steps for viral removal (such as solvent — detergent or
nano-filtration) or inactivation (such as heat). Variations to pH can also eliminate viruses.
These steps are sufficient to abolish haemagglutin activity and thus pyrogenicity.
ASSESSING MICROBIAL RISKS
Having outlined a range of microbial pyrogens, the question to ask is whether
there is a risk from microbial pyrogens that are not of endotoxin origin to pharmaceutical processes?
It is, furthermore, a problem that the various immune-stimulating components from Gram-positive
bacteria, such as lipoproteins, peptidoglycan, and lipoteichoic acids, or other pyrogens can pass the
LAL test without resulting in a signal? (20) This article argues that although such non-endotoxin
pyrogens are theoretically present, the risks that they pose are low for most processes. This is primarily
because considerable quantities of non-endotoxin microbial material are required to elicit a pyrogenic
response. For example, while staphylococcal enterotoxin is considered a "potent" pyrogen, a
concentration of 1 ug/Kg is required to elicit a pyrogenic response in the pyrogen test. When
compared to endotoxin (threshold pyrogenic dose of 0.5 EU (~ 0.05 ng/kg), the difference is 20,000
fold. Thus the likelihood of sufficient staphylococci contaminating a drug produced, under cGMP
conditions, to elicit a pyrogenic response is remote (21). The risks from these types of microorganisms
can be best assessed through bioburden monitoring. It is incumbent, however, for each
pharmaceutical manufacturer to undertake some form of risk assessment.

With the risk assessment the following were considered:

a) Types of pyrogenic substance. These were divided into those of microbial origin and non-
microbial origin.
b) Each pyrogen was considered for its association (e.g. endotoxin with Gram-negative
bacteria).
c) Each pyrogen was considered for its primary source e.g. endotoxin associated with water.
d) The likelihood of the pyrogenic substance being found in relation to manufacturing was
considered. For this the risk classes of low, medium and high were deployed.
e) Risk mitigations were considered for each pyrogen (e.g. with endotoxin this is the LAL test.)
Of these substances, however, lipopolysaccharides (endotoxins) are the most
ubiquitous and important. This is because:
Lipopolysaccharides belong to the strongest elicitors of the mammalian immune system due
to the induction of a series of cytokines such as tumor-necrosis-factor-alpha (TNFalpha) in
immunocompetent cells like mononuclear cells (22).
Where lipopolysaccharide is the specific pyrogen of concern the LAL test according to the
opinion of many authors is more sensitive in comparing with the pyrogen rabbit test (23 – 30).
Various studies carried out during the 1970s through to the 2000s found that an assortment of
samples of plasma proteins, enzymes, vaccines and blood substitutes tested comparatively in
rabbits and with the LAL test indicated that the LAL test gave similar results or was tenfold
more sensitive than the assay in rabbits
In relation to pharmaceutical processing, endotoxin is regarded as the
most likely pyrogenic substance to be found (31). Pool et al (1998) found few instances where
a lack of correlation was observed between whole blood assay, rabbit pyrogen test and LAL
(32). Therefore, the possibility of detecting pyrogens other than endotoxin from pharmaceutical
products is generally low. A separate review of blood products found, through experiments
combining polymyxin B and MAT, that pyrogenic batches were mainly contaminated with
endotoxins rather than from other types of pyrogens (33).

SOURCES OF PYROGENS IN PHARMACEUTICAL PROCESSING

Sources of pyrogens in pharmaceutical processing include solvents, drugs,
additives apparatus used in manufacture, containers. Primary sources are: the water used as the
solvent or in the processing; packaging components; the chemicals, raw materials or equipment used
in the preparation of the product. To minimize the risks from these sources, U.S. Food and Drug
Administration (FDA) recommends that there is good control of the microbiological and endotoxin
levels of contamination in the potential source (34). FDA inspection findings suggest that where
pyrogen problems were found in dosage forms, and when the source was one of the raw materials, it
was the active drug substance. This was particularly so in the case of drug substances in which process
water was used at some late stage in the synthesis process. This finding has also been supported by
correlation. Endotoxin levels of the drug substance were subsequently lowered when the
microbiological levels of the process water were lowered and the process water system was
controlled. With the manufacture of parenteral products in particular, the most usual vehicle is water

REMOVAL OF PYROGENS FROM PHARMACEUTICAL PROCESSES

Endotoxin can be inactivated when exposed at temperature of 250º C for more
than 30 minutes or 180º C for more than 3 hours (35). Acids or alkalis of at least 0.1 M strength
can also be used to destroy endotoxin in laboratory scale (36). These methods can be deployed
to depyrogenate glassware. There are a number of approaches used to reduce endotoxin
contamination of pharmaceuticals (37). These include ion-exchange chromatography, affinity
adsorbents, such as immobilized L-histidine, poly-L-lysine, poly(γ-methyl L-glutamate), and
polymyxin B, gel filtration chromatography, ultrafiltration, sucrose gradient centrifugation,
and Triton X-114 phase separation. The success of these techniques in separating LPS from
proteins is strongly dependent on the properties of the target protein. Of these methods,
ultrafiltration can be very effective at removing endotoxins from water

SUMMARY
This article has considered pyrogens of microbial origin and the
theoretical risks they pose to pharmaceutical products. The focus of the article has been with
non-endotoxin pyrogens. The reasons for this are because of the voluminous content written
about endotoxins and because non-endotoxin pyrogens often receive little discussion. Despite
the low risk posed by non-endotoxin pyrogens, questions of their occurrence and risks are
sometimes raised by regulators through inspections and by assessors during license changes
and submissions. When such questions arise the pharmaceutical manufacturer is often called
upon to undertake a risk assessment. Some of the information outlined within this article could
be helpful in developing such an assessment