Pyrogens and Depyrogenation process in sterile dosage form manufacturing

Pyrogen removal or depyrogenation

Ion exchange chromatography

Ultrafiltration

Distillation

Inactivation or destruction of Pyrogens

Acid-base hydrolysis

Oxidation

Heating

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 sterilisation 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 disscued earlier is a substance which produces fibrile conditions when it comes directly in systemic circulation.

Pyrogens are endotoxins or exotoxins , in most caseses they are endotoxins lipopolysaccharides , endotoxins from gram negative bacterias.

In human body gram negative bacterias do exit but the endotoxins produed by them are limited to certain level by human body so that a fibrile condition do not reach in normal individuals.

Test for Pyrogens:

1.Sham Test testing on rabbits

In early days all pharmaceutical industries were dependant 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 alomeration or cloting .
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:

Following are some of articles which will be useful for you in further understanding of aspects of gmp guidelines and  sterile dosage form manufacturing

Requirements of documents for validation of sterilisation process

http://whoguideline.blogspot.com/2009/11/us-fdas-requirements-of-documentation.html

How to investigate OOS out of specification results
http://whoguideline.blogspot.com/2009/09/how-to-investigate-out-of-specification.html

Determination of Phenol coeeficient of a disinfectant

http://whoguideline.blogspot.com/2009/04/determination-of-phenol-coefficient.html

Sterility testing

http://whoguideline.blogspot.com/2009/04/sterility-testing-of-pharmaceuticals.html

Clean Room Classification

Time limitations in sterile pharmaceuticals processing
http://whoguideline.blogspot.com/2010/02/time-limitations-in-aseptic-process.html

Aspects of validation of manufacturing process in sterile pharmaceuticals

http://whoguideline.blogspot.com/2010/02/aspects-of-validation-of-aseptic_26.html

Pharmaceutical Good manufacturing practices in pharmaceutical industry 

Pharmaceutical companies

What is High-Efficiency Particulate Air  HEPA Filter

What is 21 CFR Part 11, US gmp guidelines requirements of FDA compliance and CFR 21 Part 11.

FDA guidelines