2. What are pyrogens?
Pyrogens are any molecules or substances that cause a feverous reaction when
they enter the human body.
Exogenous pyrogens are molecules located outside of the body, such as
endotoxins from gram-negative bacteria or pyrogenic prions.
Exogenous pyrogens either provoke endogenous pyrogen production to create a
fever within the body or activate the body’s toll-like receptors (TLRs) to trigger a
fever.
Clinically, the fever produced by endogenous cytokines is indistinguishable from
fever produced by exogenous pyrogens such as lipopolysaccharide (LPS).
3. What is depyrogenation?
Depyrogenation is a process that removes pyrogens.
The most prevalent and problematic pyrogens are the bacterial endotoxins found in the outer
cell walls of gram-negative bacteria.
Thus, depyrogenation is a process that will either destroy or remove bacterial endotoxins.
Why needed?
Medical devices and parenteral products must be sterile and pyrogen-free. Products can
accumulate pyrogens from raw materials or other parts of the manufacturing process.
The best pyrogen removal or destruction process (known as depyrogenation) depends on the
product.
Standard depyrogenation methods are dry heat, rinsing, and filtration.
4. Depyrogenation By Dry Heat
Dry heat is commonly used for the depyrogenation of heat-stable materials.
Dry heat depyrogenation depends upon time and temperature.
Depyrogenation processes are performed at temperatures ranging from 250°C up to about 400°C.
Since bacterial endotoxins are more resistant to the effects of dry heat than bacterial spores,
depyrogenation methods also sterilize the materials they depyrogenate.
Dry heat depyrogenation uses air first to heat and then to cool items.
Due to the heat capacity of dry air, loaded objects are slowly heated and cooled during dry heat treatment.
5. Depyrogenation By Dry Heat
Items in the dry heat ovens must be placed in the
exact locations every time for depyrogenation
cycles to be valid due to the limited heat capacity of
air.
Indeed, varying load mass and product distribution
can result in dry heat processing variability.
Dry heat depyrogenation, like dry heat sterilization,
uses a combination of temperature sensors and
thermocouples to regulate temperature and dwell
time to the levels needed to kill the endotoxin load
on incoming materials.
6. Depyrogenation By Filtration
Depyrogenation filtration processes are often performed on solutions containing
proteins and peptides.
Factors to consider when selecting your depyrogenation filters are the protein
types in solution, protein concentration, electrolyte concentration, pH buffer
system, molecular weight and isoelectric point (pI) of the protein, filtration flow
rate, and protein aggregation potential.
Depyrogenation of liquids may be accomplished employing filtration through
more than one filter type.
7. Filtration Types For Sterile Product
Depyrogenation
1- Microporous Membrane Filters
Microporous membranes with pore size or retention ratings between 1.0 and 0.1 microns (μm) are
effective at removing intact bacteria via size exclusion.
Filtering freshly prepared solutions with microporous membranes can remove microbes, stop
bacterial proliferation, and prevent endotoxin formation.
Endotoxins themselves are much smaller than microporous membrane filter pores.
Indeed, endotoxins are gram-negative bacterial cell wall fragments often less than 0.025 μm in size.
However, endotoxins are negatively charged and can be removed through adsorption with
positively charged membranes.
8. cont
Endotoxin adsorption will occur with positively charged membranes until the membrane is
saturated.
Thus, it is important to change filters before saturation, or remaining endotoxins will pass
through the filter.
How well a positively charged membrane adsorbs the endotoxins is impacted by flow rate, pH,
and any surface properties of the membrane.
For example, if the flow rate is too fast for negative and positive charge interactions to occur,
the endotoxins will not be extracted from the solution.
As an alternative to positive charge adsorption, endotoxins have some adsorption affinity to
hydrophobic membranes.
9. Reverse Osmosis Membranes
Reverse osmosis (RO) membranes are the tightest size separation filters and are capable of removing
dissolved salts and sugars from water.
Essentially everything, including pyrogens, is extracted via size exclusion.
RO membranes filter best at high pressure (200–1000 psi), which allows the filter to overcome osmotic
pressure.
RO membrane filter ratings are measured by retaining or removing marker salts such as sodium chloride
or magnesium sulfate.
RO systems are often paired with downstream ultraviolet light if you wish to filter pyrogens and
microbial contaminants.
The primary risk for microbial contamination is that RO filters are used at ambient temperatures,
capable of allowing the growth of any microbiological contamination.
10. Ultrafiltration Membranes
Ultrafiltration (UF) membranes have pore sizes between one and one hundred nanometers.
These filters are traditionally rated by molecular weight cut-off (MWCO) and are made of polymeric
porous structures.
Since the endotoxin subunit, LPS, is about 10–20 kilo Daltons (kDa), membranes of 6–10 kDa MWCO are
used for size exclusion depyrogenation.
However, LPS is commonly in aggregated forms weighing 300 to 1000 kDa so that endotoxin can be
successfully removed by MWCO membranes of 30–100 kDa.
UF depyrogenation capability can be boosted using the adsorptive capabilities of hydrophobic
membranes.
Overall, UF is not recommended for depyrogenation of solutions containing large proteins but is highly
effective at removing endotoxins for small molecule drugs, buffers, electrolytes, antibiotics, and
antifungal agents.
11. Activated Carbon Depth Filters
Activated carbon is a filter adsorbent that binds colors, odors, bacterial endotoxins, and nucleic
acids.
An organic material, activated carbon, has a microstructure with millions of pores.
These pores give activated carbon its highly adsorptive properties and provide a far greater
surface area for endotoxins to interact with than polymeric microporous structures.
Activated carbon is only part of a depth filter and is often activated by steam or acid chemical
treatment.
As one of the best endotoxin removal agents, activated carbon can reduce endotoxin from
liquids by a 4-log or 5-log reduction.
However, active carbon provides nonspecific adsorption and can remove other important
elements or molecules from a liquid during filtration.
12. Pyrogen testing
Pyrogen test is designed to limit the risk of febrile reaction following the
administration of parenteral drugs.
It can be both in-vivo( Rabbit Test) and Invitro (LAL Test)
13. LAL Test
LAL-Limulus Ambeocyte lysate
LAL is an aqueous extract of blood cells (ambeocytes) from the horse shoe crab
(Limulus polyphemus).
LAL reacts with endotoxins or LPS which is the membrane component of gram
negative bacteria and forms gel which is then used for the detection and
quantification of bacterial endotoxins
14. LAL test
Types of LAL test
a. Clot formation
b. Turbidometric
c. Colorimetric
15. LAL test limitation
Disturbed by endotoxins binding components like lipids and blood components
Difficult to correlate with rabbit test
False positive for cellulose and many herbal preparations
16. Rabbit test (Sham Test)
Objective:
Is to measure the rise in body temperature of the rabbits following IV injection of
sterile solution of the substance being examined
Principle
pyrogens are the fever inducing organic substances responsible for many febrile
reactions. Pyrogen testing should be done to every batch of pharmaceutical
products (parenteral) for which water is the usual vehicle. The best animal model
for the Pyrogen test is rabbit as they generates the reproducible results that are
similar to threshold response to the humans and also economical.
17. Requirements
Animals: Healthy matured rabbits of either sex, weighing less than 1.5kg and
should be maintained at balanced diet. They should not show any sign of weight
loss during preceding week of test.
Instruments: Pyrogen free-syringes, needles, glassware. Accurate temperature
sensing device such as clinical thermometer graduated in 0.1C. Insert the
thermometer into the rectum of the rabbit not less than 6 cm and measure the
temperature.
Reagents: 0.9% NaCl injection as diluent.
18. Procedure
Select the matured healthy rabbits of either sex
House them separately in a place that is free from disturbances that likely to excite them and
maintain the room temperature 20-23C.
Making all the instruments and materials Pyrogen free either by heating for 30minutes at 25C or
any other preferred method.
Measure the rabbit temperature using a standardized clinical thermometer graduated at 0.1C.
Test them to determine that maximum reading is attaining in <5 minutes or not.
Pyrogen testing of solutions should be done in 2 steps:
a. Preliminary test (sham Test)
b. Main test
19. Preliminary test (sham Test)
Conduct the sham test using the fresh rabbits used for the first time in Pyrogen testing or not been
used during the previous 2 weeks.
Acclimatize the rabbits for 1-3 days before using for Pyrogen testing of sample.
Select 3 rabbits and fast them overnight with free access to water and withhold water during the test.
Record the temperature of the rabbits 90 minutes prior to the injection.
After 90 minutes, inject sterile Pyrogen free saline solution intravenously at a dose of 10mL/kg of the
bodyweight.
Record the temperature of the rabbit at 30 minutes interval after injection for up to 3 hours.
Exclude any rabbit that shows temperature variation of 0.6C for the main test.
This test should be performed in a room without disturbances and temperature variance must be ±3C
20. Main Test
Select 3 rabbits that passed the sham test.
Determine the initial body temperature of the rabbits and it should be between
38-39.8C.
Dilute solution with pyrogen free saline solution and warm the test liquid to 38.5C
before injection.
Inject the test solution to the rabbits slowly in the marginal vein of the ear for the
period of not more than 4 minutes and the volume injected should not be less
than 0.5mL/kg and not more than 10mL/kg of the body mass.
Determine the body temperature after every 30 minutes for 3 hours.
Endogenous pyrogens (such as the cytokine interleukin-1) are found naturally within the human body. Endogenous pyrogens create a fever-producing reaction when naturally produced by the body.
