Thursday, June 22, 2023

Pyrogens and Depyrogenation process in sterile dosage form manufacturing

Pyrogens and Depyrogenation process in sterile pharmaceuticals manufacturing

Pyrogen removal or depyrogenation

Ion exchange chromatography



Inactivation or destruction of Pyrogens

Acid-base hydrolysis



Sodium Hydroxide

Pyrogens are endotoxins and in some cases exotoxins from microbial origin , mostly from gram negative bacterias.
Pyrogens are lipopolysaccharide in conjugation with protein parts .
This is one of reason that pyrogens are not destroyed by moist heat sterilisation.
Moist heat sterilisation brings about coagulation in protein part , coagulated molecule when it comes again in contact with water regains its structure not completely but sufficient to induce antigenic response .
Dry heat sterilization is responsible for pyrolysis of pyrogenic molecule , the only ruminant then is carbon.
Pyrogen content in sterile pharmaceuticals is measured in terms of content of endotoxins.

Endotoxin levels are measured in "endotoxin units" "EU"

Following are the allowed limits for endotoxins.

Sterile dosage form Pharmaceuticals (injectable, intrathecal) - 0.2 EU/kg product

Sterile dosage form Pharmaceuticals (injectable, non-intrathecal) - 5 EU/kg product

Sterile water for injection- 0.25-0.5 EU/ml (depends on intended use)

One EU of endotoxin is approximately equivalent to 100 pg of E. Coli lipopolysaccharide the amount present in around 105 bacteria.

The process which involves removal or destruction of pyrogen in a pharmaceutical dosage form is termed as depyrogenation.

Pyrogen as discussed earlier is a substance which produces febrile conditions when it comes directly in systemic circulation.

Pyrogens are endotoxins or exotoxins , in most classes they are endotoxins lipopolysaccharides , endotoxins from gram negative bacteria's.

In human body gram negative bacteria's do exit but the endotoxins produced by them are limited to certain level by human body so that a febrile condition do not reach in normal individuals.

Test for Pyrogens:

1.Sham Test testing on rabbits

In early days all pharmaceutical industries were dependent on this test for detection of pyrogen in sterile pharmaceutical products , the test comprises of injecting substance to be tested in to albino rabbits , and observing the raise in body temperature , there is detailed method given in pharmacopoeia's .

What is the advantage of this is that , it provides you exact evaluation presence of pyrogen. In LAL test the antibodies of LAL reagent are capable of reacting with only gram negative endotoxins , still it is most reliable and valuable test in detection and quantification of endotoxins in a sterile dosage form.

2.LAL Test :Limulus Amebocyte Lysate

This test is widely used in all over the world and it is most reliable too ,
In this test employs antigen derived from blood of horseshoe crab , this antigen reacts with endotoxins and form agglomeration or clotting .
This test is very sensitive and can detect endotoxin up to 0.005 EU/ml
Following are applications of LAL tests: formation of gel clot, turbidimetric estimation, colorimetric, and chromogenic assay:

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