1. what is pyrogen in sterile preparation?

2. why the sterile preparation must be pyrogen-free?
3. how to make preparation with pyrogen-free (how to test)?
4. is something sterilized, its pyrogen-free?
5. how to test your sterile preparation is pyrogen-free? (15m)
6. test for lab scale(how to test the preparation is pyrogen-free)?

Sterile vs. pyrogen-free – it’s important to your work and you need to know
the difference. First of all, what is a pyrogen? Simply, a pyrogen is any
substance that causes a fever. The most commonly thought of pyrogens are
bacterial endotoxins and exotoxins, although the host body (human or animal)
can also produce pyrogens. The most commonly assayed for pyrogen is
lipopolysaccharide, or LPS. It is a component of the bacterial wall of Gram-
negative bacteria and is released upon breakdown of the cell wall or bacterial
cell lysis.
So, if something has been sterilized, it should be pyrogen-free right? Wrong!

WHEATON CryoELITE in BenchMate Rack
Sterility ensures the absence of viable living bacteria. However, the act of
sterilizing a contaminated vial can actually result in the release and deposit of
pyrogens, as sterilization destroys bacteria, leading to bacterial cell lysis and
release of LPS or other endotoxins and exotoxins.
Make sure your products are manufactured in a pyrogen-free setting that
prevents the deposition of bacteria and other agents on the products.
Required sterile products would then undergo a separate sterilization process
dependent on the product and/or end-users’ needs. Thus, specific items may
be sterile, sterile and pyrogen-free, or pyrogen-free but not sterile.
Thus, it’s important to consider your specific needs in order to ensure that
your needs are met by your product of choice.
Key Points:
1) Sterile does not mean pyrogen-free.
2) Manufacturing that takes into account pyrogen-free environments are
best.

Pyrogen testing defines a process used by drug manufacturers to determine if
bacterial toxins are present in vaccines and drugs that might cause fever when
used on humans. It determines if microbes or their metabolites are present in
intravenous solutions during the manufacturing process. The most common
and oldest form of pyrogen testing consists of injecting drugs into rabbits to
determine if a fever develops. A newer test uses blood from the horseshoe
crab to test for toxins.
The rabbit pyrogen testing method surfaced in the 1940s after some patients
became ill from intravenous drugs. Hypodermic devices at the time proved
useful for administering drugs directly into the bloodstream for patients who
were unable to tolerate oral medications. Even though hypodermics devices
were sterile, the drugs were not always safe.
Patients sometimes developed high fevers, chills, and body aches, and some
people suffered shock. Doctors didn’t know why this occurred, frequently
calling the condition injection fever, saline fever, or distilled water fever.
Researchers later discovered some drugs and vaccines were contaminated in
production labs with pyrogen endotoxins, potent bacteria that withstand
sterilization.
The human body fights exposure to bacterial toxins in the environment
through the skin. When contaminated drugs are injected into the bloodstream,
toxins bypass normal defense mechanisms. White blood cells begin releasing
another form of pyrogen that causes high fever, which might lead to shock and
death.
Pyrogen testing in drug labs involves heating equipment used to ensure
sterilization. The drug is injected into ear veins of rabbits to see if a fever
develops. Rectal temperatures of test animals are analyzed after 30 minutes
and again one to three hours later. If the animals remain fever-free, the
solution is free from toxins.
The process of pyrogen testing on animals typically involves injecting several
rabbits at a time within a 10-minute time frame. The dosage for each rabbit
depends on body weight, age, and gender. The same group of rabbits might be
tested repeatedly every few days until they develop a tolerance to the drugs.
A newer pyrogen testing technique is called the limulus amoebocyte lysate
(LAL) test. Blood from horseshoe crabs contains high levels of toxins naturally
found in marine life. Scientists discovered a way to use the blood to test for
bacterial toxin in drugs and the raw materials used to manufacture medicine.
The LAL pyrogen testing procedure might be 100 times more sensitive than the
rabbit testing methods. Medical devices that are implanted into humans go
through LAL testing, along with radioactive drugs and anesthesia.

Rabbit Pyrogen Test
A pyrogen is a foreign substance that causes a fever (temperature elevation) in
an animal’s body. Typically, pyrogenic substances include endotoxin and other
bacterial byproducts. Vaccines and other injectable drugs must be confirmed
to be pyrogen free according to regulatory requirements of 21CFR, USP, and EP.
The typical assay for endotoxin contamination detection is the LAL test. In
some cases, the LAL test may not be feasible. These cases might include:

 Interference by the test article
 The presence of a compound capable of neutralizing endotoxins
 The presence of pyrogenic substances other than endotoxin,
 Certain regulatory requirements

BioReliance offers the rabbit pyrogen test, in addition to the LAL test, as an
alternative assay for the detection of endotoxin and other pyrogens. The rabbit
pyrogen test requires the injection of a small amount of batched test material
into a rabbit’s blood stream, and monitoring for temperature increases.

ENDOTOXIN (LAL) TESTS

BACKGROUND:
Endotoxins are bacterial structural components that are released when such a
cell is lysed. These components are toxic if administered to humans and/or
animals, causing a pyrogenic response (rise in body temperature). For this
reason it is important that drugs and medical devices which are either injected
or implanted must be tested for their endotoxin content. There are several
methods available for conducting the endotoxin test, which includes the in
vivo rabbit pyrogen test and several in vitro alternatives that utilize the Limulus
Amebocyte Lysate (LAL) system. The latter has become the method of choice,
which can be accomolished by various options including gel clot, kinetic
chromogenic and kinetic turbidetric assays. This methodology is also used for
the evaluation of medical devices such single- use disposable equipments and
implants. This is done by extracting the test product with pyrogen -free water
(PFW) and testing for the presence of endotoxin in the extracts.

PRINCIPLE:
Kinetic Chromogenic method and Kinetic Turbidimetric method
ams Laboratories are able to perform both the kinetic chromogenic and
turbidimetric assays. The chromogenic method involves an enzymatic reaction
between the endotoxin and lysate which results in the production of a yellow
colour in the presence of endotoxin. The intensity of the colour production is
directly linked to the quantity of endotoxin present in the sample. With the
kinetic variation of the assay, the time of onset of the colour reaction is
measured. Therefore, with the use of endotoxin standards we are able to
calculate the value of endotoxin present in or on the product. Some products
are of a colour that would interfere with this form of testing, and so the
turbidimetric method can be used to avoid any such interference. In this case a
different lysate is used and the reaction with endotoxin results in the solution
becoming turbid, thus allowing quantitation of endotoxin content without
relying on the colour present. Both methods are equally effective in obtaining
the endotoxin content in a product, but often, one is more suitable than the
other. Both methods use objective measurements to determine endotoxin
content and are quantitative in nature. These tests can be carried out
relatively quickly, and results can be available within 3-5 days of sample receipt.
Gel Clot Assay method
The gel clot assay was the original LAL method and relies upon the operator to
distinguish the formation of the gel clot in the reaction tubes. It is a qualitative
or semi-quantitative test that is used to screen for the presence of endotoxins.
A clot formation is interpreted as a positive result for the presence of
endotoxin and if no clot forms, this is interpreted as the sample being
endotoxin free. The results are from the subjective interpretation of the clot
formation.

SAMPLE REQUIREMENTS AND TURNAROUND TIMES:
Generally one container is sufficient for method qualification
purposes. For validation of a new product, the pharmacopoeias require
samples from three separate batches to be tested to ensure any batch-to-
batch variation is taken into account.
Routine test results can be completed in 2 – 3 working days. Depending on
work loads in the laboratory, validations can be completed in 4 – 5 working
days.


Sterility Testing
Sterility tests are applied to products intended to be sterile before
marketing (e.g. ophthalmic and parenteral preparations) to check that these
products are free from all living microorganisms. Sterility tests are performed
on random samples from the batch and must be carried out under aseptic
conditions in order to avoid accidental contamination of the product during
the test using, for example, a laminar air flow cabinet.
Control Tests:
Two types of control tests must be performed exactly under the same working
conditions as the test.
1. Positive controls:
They are essential to show that the m.o. will actually grow under the
conditions of the test.
2. Negative controls:
These are uninoculated tubes of each media to confirm its sterility.
Methods of Sterility Test:
1. Direct inoculation of culture media:
This test is performed by direct transferring of the preparation into the
culture media. However, in case of large volumes such as intravenous
fluids, a concentrated medium may be added directly to the preparation
in its container.
2. Membrane filtration method:
By filtering the sample aseptically through a membrane filter, then
inoculate the membrane on appropriate culture media.
Application of sterility test to some dosage forms
1. Aqueous solutions: directly tested by direct inoculation method if it is of
small volume or by membrane filtration method if it is of large volume.
2. Soluble solids: dissolve in a suitable solvent that is sterile and has no
antimicrobial activity.
3. Oils and oily solutions:
Oils of sufficiently low viscosity are filtered directly via a dry
membrane filter.
Viscous oils are diluted first with suitable sterile diluent. When
the direct inoculation method is used, add a suitable emulsifying
agent that is sterile and has no antimicrobial activity. During
incubation, oily preparations should be shaken gently every day.
4. Ointments and creams (fatty base or w/o emulsions): dilute with a
suitable diluent that is sterile and has no antimicrobial activity. Add a
suitable emulsifying agent to the media in case of using direct
inoculation method.
Inactivation of Inhibitory Agents:
The presence of inhibitory agents in the preparation tested prevents
the growth of contaminating m.o. by its antimicrobial action.
Inactivation of antimicrobial agents can be carried out either by:
1. Dilution of the preparation to less than MIC (minimum inhibitory
concentration) of the antimicrobial agent.
2. Addition of inactivating agent into the culture media.
e.g. inactivation of sodium benzyl penicillin by addition of penicillinase
solution.
Inactivation of sulphathiazole by addition of 0.005% para amino
benzoic acid.
Pyrogen
Pyrogen is products of the growth of m.o. It may be parts of dead cells of a
m.o. or metabolic products.
Pyrogen chemically is lipid in nature, sometimes containing phosphorous and is
attached to polysaccharide or protein or both.
The presence of pyrogen in pharmaceutical product causes a febrile reaction in
human beings. Other symptoms like chills, pain in back and legs, and malaise
may occur. Pyrogen is rarely fatal.
Sources of Pyrogen:
Pyrogen is produced in a product either by:
1. Water used in the preparation of the pharmaceutical product, so
water must be properly distilled before use.
2. Equipments used in the preparation as pyrogen adheres strongly to
the glass and other surfaces.
3. The solute, as pyrogen may be trapped within the particle layers of
the solute during its crystallization or precipitation from aqueous
solutions containing pyrogenic contamination. In such cases, the solute
must be purified by recrystallization.
Elimination of Pyrogen:
Pyrogen can be destroyed by:
1. Heating at high temperature:
Usually heating to 250 °C for 45 min or at 650 °C for one min is
sufficient to destroy pyrogen.
This method is suitable for glasses and other metal equipments.
However, plastic materials cannot be treated by heating. It is better
to wash them with detergents.
Autoclaving cycle cannot destroy pyrogen.
2. Distillation:
It is the most suitable method for removal of pyrogen from water as
pyrogen is not volatile, so it will remain in the container.
3. Adsorption:
This method is used for solutions that cannot be distilled. Pyrogen
can be adsorbed on the surface of adsorptive agent.
4. Other useful methods used for elimination of pyrogen are: heating
with dilute alkali, dilute acid, or mild oxidizing agent.
Test for pyrogen:
Parenteral solutions must be tested for the presence of pyrogen by a
biological test in which the fever response of rabbits is used as the criteria.
Animal used:
Healthy, mature rabbits of the same weight and size are used (their
body weights must by maintained during the experiment).
Temperature recording:
An accurate temperature device must be used for this test. The device is
inserted in the rectum of the rabbit to a depth of 1.5 cm for sufficient time
(~ 3 min).
Procedure:
1. In the day of the test the food is withheld until the completion of
the test.
2. The initial temp. of the animal must be recorded and rabbits with
temp. more than 39.8 C are excluded.
3. Syringes, needles and all glassware used must be pyrogen free.
4. The sample is warmed before the injection.
5. The sample is injected into the ear vein of the rabbit (group of three
rabbits). The dose is 10 mL per kg of body weight.
6. Measure the temp. after 30 min.
7. The temp. is again determined after 1, 2 and 3 hr after the injection.
8. The test is positive when each rabbit shows an increase in temp.
more than
0.6 ºC or the sum of temp. increase of the 3 rabbits exceeds 1.4 ºC.
9. The test must be repeated if only 2 rabbits of the 3 show increase in
temp. In the repeated test, use group of 5 rabbits and the test is
positive if 4 of the rabbits show increase in temp.
Preparing pyrogen-free water using ultrafiltration

Ultrafiltration is a membrane-based technology that removes endotoxins by a
filtration process. Ultrafilters are depth filters with nominal weight cut-off
ranging from 5 000 Da to 100 000 Da. To remove endotoxins, filters with a
molecular weight from 5 KDa to 13 KDa are selected.

Ultrafilters are positioned at the point-of-use, at the outlet of the water
purification process. The water delivered is pyrogen-free and does not require
autoclaving.

Data shown on Figure 2 demonstrate the efficiency of endotoxin removal by a
13 KDa ultrafilter (BioPak®). This ultrafiltration device is fully efficient over a
four month period and large quantities of pyrogens.



Demonstration of endotoxin removal by a 13 KDa ultrafilter (BioPak)


Conclusions
Ultrafiltration is an efficient and simple way to obtain pyrogen-free water on-
demand. It avoids autoclaving procedures on water. Pyrogen-water purified
using ultrafiltration can be utilized safely for cell culture, including stem cell
culture.

The BioPak ultrafilter comes with a certificate guaranteeing endotoxin-free
water for four months when a BioPak point-of-use filter is connected to a high
purity water purification system, and utilized in normal laboratory conditions.