Pyrogens

Pharmaceutical Microbiology 2

Dr. Mohammad Abu Sini

Al-Zaytoonah University of Jordan
mohammad.abusini@zuj.edu.jo

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 Nature of endotoxins
• Endotoxin: a pyrogenic (fever inducing) substance (e.g. lipopolysaccharide) present in the
bacterial cell wall.

• Endotoxins are composed of three subunits: the inner lipid A is linked to a central
polysaccharide core that is joined to long projections known as the O-antigenic side chain.

• Lipid A is responsible for most of the biological activity of endotoxin.

• The human body’s reaction to endotoxins range from fever to death.

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Nature of endotoxins
 Known Endotoxin Areas
• Elevated airborne concentrations are prevalent in:

1. Sewage treatment plants

2. Swine operations

3. Cotton textile mills

4. Poultry houses

5. Water damaged buildings or in the presence

of a contaminated humidifier
 Biological activity of pyrogens

• At low dose levels endotoxin will:

1. Directly affects the thermoregulatory centers in the brain.

2. Produces airway inflammation (chest tightness & wheezing).

3. Associated with increased severity of child and adult asthma.

4. Exposure to endotoxins during childhood may reduce allergic responses later in life.

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 Biological activity of pyrogens

• At high dose levels endotoxin will:

1. Activate the coagulation system

2. Alter carbohydrate and lipid metabolism

3. Produce platelet aggregation

4. Produce shock and then death

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 Biological activity of pyrogens
• Other sources of pyrogenic substances include:

1. Peptidoglycan of Gram-positive bacteria.

2. Exotoxins of Gram positive; erythrogenic response produced by Streptococcus group A.

3. Viruses, common cold virus induce a pyrogenic response and fever.

4. Molds and yeasts also produce a pyrogenic effect following intravenous injection.

5. Non-microbial pyrogens, such as steroids and plasma components.

• Nonetheless, the most important pyrogens in pharmacy products are high molecular weight
endotoxins that are found in the outer membrane of Gram-negative bacteria (LPS).
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 Laboratory Analysis of Endotoxins
• Bacterial endotoxin test: is a specific test for endotoxins of bacterial origin.

• Bacterial endotoxin test is commonly referred to as the limulus amoebocyte lysate (LAL) test.

• This test allows the use of a lysate of amoebocytes from either the American or Japanese horse-
shoe crab.

• Rabbit test was not allowed to be used in the BP in 2002.

• All equipment and reagents used in test must be endotoxin free.
• Validation of accuracy and reliability of the used LAL method for
each product is essential.

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• In the blood cells of the Atlantic horseshoe crab, Limulus polyphemus, there are mobile cells
called amoebocytes.

• Amoebocytes contain granules with a clotting factor known as coagulogen.

• Coagulogen is released outside the cell when bacterial endotoxin is encountered.

• The resulting coagulation is thought to neutralize endotoxins and to contain bacterial infections
in the animal’s circulation.
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Laboratory Analysis of Endotoxins
• Types of LAL test accepted by the International Pharmacopeias:

1. Gel Clot (Limit Test)

2. Gel Clot (Semi-Quantitative test)

3. Turbidimetric Kinetic Method

4. Chromogenic Kinetic Method

5. Turbidimetric End point Method

6. Kinetic End point Method

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 Endotoxin Testing . . . The Gel Clot Method
• The original method, or the official “referee test”.

• This is an endpoint method.

• The specimen is incubated with LAL of a known

sensitivity.

• Formation of a gel clot is positive for endotoxin.

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 Endotoxin Testing . . . The Turbidimetric Method
• A kinetic method.

• The specimen is incubated with LAL.

• Either the rate of increase in turbidity or the

time taken to reach a particular turbidity is
measured spectrophotometrically and
compared to a standard curve.

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 Endotoxin Testing . . . The Colorimetric Method
• Can be kinetic or endpoint method.

• Endotoxin catalyzes the activation of a

proenzyme in LAL, which will cleave a
colorless substrate to produce a colored end-
product.

• The colored end-product can be measured

spectrophotmetrically and compared to a
standard curve.

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 Depyrogenation
• Depyrogenation is the elimination of all pyrogens, from the production materials, solutions and
equipment.

• The main method of preventing pyrogens contaminating parenteral products is strict control of
the ingredients used.

• Ingredients of parenteral products (solvents, raw materials, packaging materials and equipment)
should be free of pyrogens.

• This is achieved by either removal or inactivation of the pyrogens.

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Depyrogenation
• To inactivate pyrogens various methods are used:

1. Heat treatment

2. Distillation

3. Ultrafiltration

4. Acid-base hydrolysis

5. Oxidation

6. Rinsing

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Depyrogenation by heat treatment
• Endotoxins and extremely heat stable; hence high temperature is widely used to incinerate
pyrogens especially for glassware, thermostable equipment, and formulation components.

• The commonly used dry or moist heat sterilization cycles will not greatly reduce the pyrogen
burden of parenteral products.

• The generally recommended inactivation condition is dry heating at 180°C for 3 hrs., or dry-
heat at 250°C for 30 minutes.

• A simple method of removing small amounts of pyrogens from surfaces such as packaging
components is by rinsing the surfaces with non-pyrogenic water.

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 Depyrogenation of water
• Endotoxins are water soluble; therefore, water is the greatest source of pyrogens in parenteral
products.

• Untreated water is considered contaminated with pyrogens, therefore the removal of

endotoxins (depyrogenation) must be practiced in water used in parenteral products.

• Contaminated solutions will become more pyrogenic with the passage of time. Therefore,
these products must be sterilized shortly after preparation.

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 Depyrogenation of water

• As pyrogens are non-volatile, distillation is the principal method of avoiding contamination of

water used in parenteral products.

• The freshly collected distillate, which is initially pyrogen-free water, can become contaminated
with organisms and pyrogens if stored for more than 4 hours at 22°C.

• To avoid microbial growth in this water it must be sterilized soon after collection or stored at
high temperature to suppress microbial growth.

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 Depyrogenation of water

• Pyrogens can also be removed from solutions by ultrafiltration.

• Ultrafiltration separates pyrogens by a process based on their relative molecular mass.

• The filters used in ultrafiltration are different from the 0.22 µm filters often used in pharmacy
production.

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