Test for pyrogen and various methods to determine pyrogenicity

2. Pyrogen
• Pyrogens, a chemically
heterogeneous group of fever-
inducing compounds, originate
from bacteria, viruses, fungi or
the host itself

3. • Most common pyrogens which are of
significant in pharmaceutical industry are
Gram negative bacterial endotoxin
• Endotoxin are part of the outer membrane
of the cell wall of gram negative bacteria
• Bacteria constantly shed endotoxin into
environment as they grow & multiply ,as
well as when they die & disintegrate

4. Source of pyrogen
• Water
• Packaging components
• Chemicals & raw materials
• Equipment used in the preparation of the
product.
• The presence of endotoxins on devices may
be attributed to water in
the manufacturing process.
• Washing of components such as filter media
(filters) ,rinsing of tubing or other plastic
devices prior to their sterilization

5. Classification of Pyrogens
• Endogenous pyrogen is a low-molecular-weight protein that is
produced by phagocytic leukocytes in response to stimulation by
exogenous pyrogens and released into the circulation. It induces fever
by acting on the preoptic area of the hypothalamus to raise the set-
point of the hypothalamic thermostat
• Exogenous pyrogen is a fever-producing agents of external origin, e.g.,
bacterial endotoxins and other microbial products, antigen-antibody
complexes, viruses and synthetic polynucleotides, incompatible blood
and blood products, and androgen breakdown products such as
etiocholanolone

6. Endotoxin characteristic
• Thermostable
• Water-soluble
• Unaffected by the common bactericides
• Non-volatile
These are the reasons why pyrogens are difficult to destroy once
produced in a product

7. Pyrogenicity
Ability to cause a change in body temperature.
The active chemical agent that causes the
temperature change is an endogenous pyrogen that
is released from the blood leukocytes under the
influence of the endotoxins
The pyrogen affects the hypothalamus of the brain,
which regulates body temperature

9. Test for Pyrogen

10. RPT

11. LAL
• Horseshoe crabs have a primitive immune system, so they fight off
infection with a compound in their blood called Limulus Amebocyte
Lysate. LAL binds and clots around fungi, viruses and bacterial
endotoxins, protecting the crabs from infection.

12. Fred Bang reported in 1956 that gram-negative bacteria, even if killed,
will cause the blood of the horseshoe crab to turn into a semi-solid
mass. It was later recognized that the animal's blood cells, mobile cells
called amoebocytes, contain granules with a clotting factor known as
coagulogen; this is released outside the cell when bacterial endotoxin is
encountered. The resulting coagulation is thought to contain bacterial
infections in the animal's semi-closed circulatory system

13. Monocyte activation Test

14. RPT vs LAL vs MAT

16. Pyrogen removal and inactivation or Destruction
• Ion exchange chromatography
• Ultrafiltration
• Distillation
• Reveres Osmosis
• Dilution or rinsing
• Adsorption
• Chemical Sterilization & Gaseous sterilization
• Ionizing radiation
• Moist heat & Dry heat

17. • https://www.sigmaaldrich.com/technical-
documents/articles/microbiology/monocyte-activation-test-for-
pyrogen-detection.html